Compositions and methods for targeted cytokine delivery

ABSTRACT

The present disclosure encompasses compositions and methods for targeted cytokine delivery. The compositions disclosed herein comprise a cytokine linked to a ligand and may improve immunotherapy by limiting side effects associated with immunotherapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. application Ser. No.15/536,580, filed Jun. 15, 2017 which claims the benefit of PCTApplication PCT/US2015/065872, filed Dec. 15, 2015, which claims thebenefit of U.S. Provisional Application No. 62/243,829, filed Oct. 20,2015 and U.S. Provisional Application No. 62/091,898, filed Dec. 15,2014, each of the disclosures of which is hereby incorporated byreference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under A1073552,HHSN27220070058C, and HL113931 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure encompasses compositions and methods for targetedcytokine delivery. Through specific delivery of cytokines, thecompositions disclosed herein may improve immunotherapy by limiting sideeffects associated with immunotherapy.

BACKGROUND OF THE INVENTION

Systemic administration of high dose interleukin 2 (IL2) is one of themost potent forms of immunotherapy and is currently approved by the FDAfor treatment of several malignancies. Efficacy of this treatmentdepends on activating cytotoxic lymphocytes (CTLs) such as naturalkiller cells (NK) and CD8⁺ T lymphocytes (CD8⁺ CTLs). Clinical trialshave demonstrated approximately 15% partial or complete tumor responses,with up to 5% of patients having a durable long-lasting responseresembling a cure. Despite these encouraging results in a minority ofpatients, most do not achieve a benefit or stop IL2 therapy prematurelydue to complications such as blood pressure changes and pulmonary orsystemic capillary leak. It is thought that the direct action of IL2 onvascular endothelium contributes to the majority of these side effects.The efficacy of IL2 is also limited by preferential activation ofCD4⁺Foxp3⁺ regulatory T cells (T_(regs)), which decrease the tumorimmune response. For these reasons treatment with high-dose IL2 hasfallen out of favor clinically.

Side effects and deceased efficacy of IL2 therapy occur due to the highaffinity trimeric αβγ IL2 receptor (IL2R), which is expressed byvascular endothelial cells and T_(regs) at baseline. Thus CD4⁺Foxp3⁺T_(regs) and vascular endothelium are activated at much lower doses ofIL2 than NK cells, which express the lower affinity βγ chains of theIL2R at rest. NK cells do express the high affinity α chain of IL2Rafter activation and depend on this trimeric receptor for peak cytolyticcapacity. Mutant forms of IL2 with decreased affinity for IL2Rα havebeen described and offer a more favorable side effect profile. However,they also result in lower efficacy and decreased therapeutic potentialdue to decreased CTL activation. Therefore, there is a need in the artfor a form of IL2 that could preferentially bind to and activate CTLswithout activating T_(regs) and endothelial cells. Such an IL2derivative might overcome such clinical barriers and result in moreefficacious immunotherapy with fewer side effects.

SUMMARY OF THE INVENTION

In an aspect the disclosure provides, a composition comprising acytokine linked to a ligand, wherein the ligand is not a correspondingbinding partner to the cytokine.

In another aspect, the disclosure provides a method to deliver acytokine to a target cell comprising contacting a target cell with acomposition comprising a cytokine linked to a ligand. The ligandspecifically binds to a receptor on the target cell and the ligand isnot a corresponding binding partner to the cytokine.

In still another aspect, the disclosure provides a method to activateimmune cells comprising contacting an immune cell with a compositioncomprising a proinflammatory cytokine linked to a ligand. The ligandspecifically binds to a receptor on the immune cell thereby activatingthe cell and the ligand is not a corresponding binding partner to thecytokine.

In still yet another aspect, the disclosure provides a method to treat atumor comprising identifying a subject with a tumor and administering tothe subject a therapeutically effective amount of a compositioncomprising a proinflammatory cytokine linked to a ligand. The ligandspecifically binds to a receptor on a target cell and the ligand is nota corresponding binding partner to the cytokine.

In a different aspect, the disclosure provides a method to treat a viralinfection comprising administering to the subject a therapeuticallyeffective amount of a composition comprising a proinflammatory cytokinelinked to a ligand. The ligand specifically binds to a receptor on atarget cell and the ligand is not a corresponding binding partner to thecytokine.

In other aspects, the disclosure provides a chimeric peptide comprisinga cytokine peptide and a ligand peptide. The cytokine peptide is not abinding partner of the ligand peptide.

In certain aspects, the disclosure provides a chimeric peptidecomprising a cytokine peptide and a ligand peptide. The ligand peptidebinds to an NKG2D receptor.

In another different aspect, the disclosure provides a nucleic acidmolecule comprising a sequence encoding a chimeric peptide of thedisclosure.

In yet another different aspect, the disclosure provides apharmaceutical composition comprising a chimeric peptide of thedisclosure.

In still yet another different aspect, the disclosure provides a methodof treating a subject diagnosed with cancer comprising administering tothe subject a pharmaceutical composition of the disclosure.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E and FIG. 1F depict adiagram, immunoblot and graphs showing the generation and in vitroevaluation of OMCP-mutIL-2. (FIG. 1A) Schematic structure ofOMCP-mutIL-2. (FIG. 1B) Molecular weight of OMCP-mutIL-2 compared tomutIL-2 and wild-type IL-2. IL-2, mutIL-2, and OMCP-mutIL-2 wereproduced in mammalian cells and have higher molecular weights due toglycosylation. The lower migrating band for mutIL-2 corresponds tounglycosylated protein, likely due to lysis of the producing cells.Based on differences in molecular weight all cytokines and constructwere administered on a molar basis with 1 μl of 4.4 μM solution definedas 1000 IU equivalents (IUe) herein. This effectively allows forequimolar comparison between IL-2, mutIL-2 and OMCP-mutIL-2 despitedifferent molecular weights. (FIG. 1C, FIG. 10) In vitro activation ofA/J lymphocyte subsets after 36 hours of culture in 100 IUe of cytokinesor OMCP-mutIL-2 construct. (FIG. 1E, FIG. 1F) Proliferation of B6lymphocyte subsets after 5-day culture in 1000 IUe/ml of cytokines orOMCP-mutIL-2 construct. Graphs representative of 3-6 replicates percondition. black=saline; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2.

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, FIG. 2F, FIG. 2G, FIG. 2H,FIG. 2I, FIG. 2J, FIG. 2K, FIG. 2L, FIG. 2M, FIG. 2N and FIG. 2O depictgraphs and images showing in vivo dosing of IL-2 and IL-2 constructs.Animal mortality (FIG. 2A) and morbidity assessed by weight loss (FIG.2B) accumulation of ascites and pleural fluid (representativesyringe-FIG. 2C; average from all mice in the group-FIG. 2D) and (FIG.2E) organ inflammation after administration of wtIL-2. Animal mortality(FIG. 2F, FIG. 2H, FIG. 2J) and morbidity as assessed by weight loss(FIG. 2G, FIG. 2I, FIG. 2K) after administration of high dose wtIL-2(FIG. 2F, FIG. 2G), OMCP-mutIL-2 (FIG. 2H, FIG. 2I) and mutIL-2 (FIG.2J, FIG. 2K) in anti-AsialoGM1 (solid line) or rabbit IgG-treated(dotted line) in A/J mice. Weight loss (FIG. 2L), ascites(representative syringe-FIG. 2M; average from all mice in the group-FIG.2N) and organ inflammation (FIG. 2O) in mice treated with 200,000 IUe ofeither wt IL-2, OMCP-mutIL-2 or mutIL-2. All graphs represent 46 animalsper treatment condition. ns p>0.05; * p<0.05; ** p<0.01; *** p<0.001;black=saline; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3H,FIG. 3I and FIG. 3J depict graphs and images showing immunologic changesassociated with IL-2 and IL-2 construct administration in vivo. (FIG.3A, FIG. 3B) Total splenocyte counts after a five-day course of 200,000IUe of IL-2 (blue), mutIL-2 (green) and OMCP-mutIL-2 (red). (FIG. 3C) NKcell expansion and activation after IL-2, mutIL-2, OMCP-mutIL-2, highdose IL-2, high dose mutIL-2 and IL-2/anti-IL-2 complexes measured bycell counts in the spleen (top) and KLRG1 upregulation (bottom). (FIG.3D) CD4⁺Foxp3+T_(reg) expansion and activation as measured by cellcounts in the spleen (top) and ICOS upregulation (bottom) as well as(FIG. 3E) NK/T_(reg) ratio in the spleen. Expansion of splenocytes (FIG.3F, FIG. 3G) and NK cells (FIG. 3H) in B6 mice treated with 750,000 IUeof cytokine or construct. T_(reg) expansion and activation (FIG. 3I) aswell as NK:T_(reg) ratio (FIG. 3J) in the spleen of B6 mice. All graphsrepresent an average cell count ±SEM from 5-10 mice per group. nsp>0.05; * p<0.05; ** p<0.01; ***p<0.001; black=saline; blue=wtIL-2,red=OMCP-mutIL-2, green=mutIL-2.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D and FIG. 4E depict graphs and imagesshowing cytokine-mediated tumor immunotherapy. (FIG. 4A) In vivocytotoxicity for YAC-1 lymphoma after intravenous injection. (FIG. 4B,FIG. 4C) LLC tumor growth after a five-day course of 750,000 IUe ofcytokine treatment given as ten doses on days 5-10 post tumor injection.LLC tumor growth in mice depleted on NK cells (FIG. 4D) or mutant micedeficient in NKG2D (FIG. 4E). Data represents 5-6 mice per group. nsp>0.05; * p<0.05; ** p<0.01; *** p<0.001; black=saline; blue=wtIL-2,red=OMCP-mutIL-2, green=mutIL-2.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H,FIG. 5I, FIG. 5J and FIG. 5K depict graphs and a schematic showing IL-2signaling in NK cells. (FIG. 5A, FIG. 5B) Serum levels after injectionof 1×10⁶ IUe of fluorochrome-labeled cytokine or construct i.v. (FIG.5C) Degranulation of NK cells in the presence of cytokines andpentameric OMCP-mediated crosslinking of NKG2D as measured by surfaceCD107a expression at 1000 IUe/ml. STAT5 phosphorylation in isolated NKcells from A/J (FIG. 5D) or B6 mice (FIG. 5E) by increasing doses ofcytokine. Decay in STAT5 phosphorylation after a 15 minute stimulationby 1000 IUe/ml (FIG. 5F) or 100 IUe/ml (FIG. 5G) of IL-2 orOMCP-mutIL-2. (FIG. 5H) Proposed model of competition between NK cellsand stromal cells for IL-2. (FIG. 5I) STAT5 phosphorylation of B6 NKcells in the presence of other splenocytes by wtIL-2 and OMCP-mutIL-2.(FIG. 5J) STAT5 phosphorylation of wild-type or NKG2D^(−/−) NK cells bywtIL-2 and OMCP-mutIL-2 in the presence of competing splenocytes. (FIG.5K) STAT5 phosphorylation, as measured by fold change oversaline-treated controls, of wild-type NK cells in the presence ofcompeting splenocytes treated with saturating concentrations of ratanti-mouse CD25 (clone 3C7) or rat IgG isotype control.

FIG. 6 depicts graphs showing B6 NK cells are preferentially activatedby low dose OMCP-mutIL-2 but this selectivity disappears at the highestdoses of cytokine or in the absence of NKG2D expression by NK cells.Left two graphs show B6 NK cells and right two graphs show BKNKG2D^(−/−) NK cells.

FIG. 7A, FIG. 7B and FIG. 7C depict imaging showing that inspection ofthe viscera demonstrates limited food consumption after a 5-day courseof 200,000 or 750,000 IUe of wtIL-2. FIG. 7D depicts a graph showingthat unlike the A/J strain, B6 mice are able to tolerate higher doses ofwtIL-2 with only moderate weight loss after 750,000 IUe. Higher doses of1,500,000 IUe IL-2 resulted in increased weight loss. Doses above thisregimen led to animal death.

FIG. 8A depicts a graph showing that A/J mice treated withIL-2/anti-IL-2 antibodies or high dose mutIL-2 lost significant weightduring treatment. The majority of IL-2/anti-IL-2 treated mice could notsurvive the full 200,000 IUe dosing and were sacrificed four days afterstarting treatment thus receiving 160,000-180,000 IUe. FIG. 8B depicts agraph and flow cytometric plot showing NK expansion with ULBP3-mutIL-2and lower doses of OMCP-mutIL-2 in A/J spleen (top). NK activation, asmeasured by surface KLRG1 expression on NK cells treated with 200,000IUe of mutIL-2 (green) or ULBP3-mutIL-2 (purple) in A/J spleen (bottom).FIG. 8C and FIG. 8D depict graphs showing that unlike the case for NKcells, little expansion of CD8⁺ or CD4⁺ Foxp3⁻ T cells was evident ineither IL-2, OMCP-mut-IL-2, or mutIL-2 treated mice. FIG. 8E depicts agraph showing weight loss in B6 mice treated with high dose mutIL-2 orIL2/anti-IL-2 antibody complex. FIG. 8F and FIG. 8G depict graphsshowing expansion of CD8⁺ or CD4⁺Foxp3⁻ T cells in cytokine treated B6mice. Graphs represent 5-10 mice per group.

FIG. 9A and FIG. 9B depict graphs showing in vitro lysis of A/J tumors,such as LM2 lung adenocarcinoma (FIG. 9A) or YAC-1 lymphoma (FIG. 9B) bybulk splenocytes after a five day course of 200,000 IUe of cytokinegiven over ten doses. FIG. 9C shows in vitro lysis of LLC lung cancer byB6 splenocytes treated with 750,000 IUe of cytokines or constructs givenover five days in ten doses.

FIG. 10A depicts flow cytometric plots showing that plate boundanti-NKG2D antibody (clone A10)-mediated augmentation of NKdegranulation with cytokines added at 1000 IUe/ml. FIG. 10B depicts aflow cytometric plot showing CD69 levels on NK cells cultured at 100IUe/ml of OMCP-mut-IL2 or mutIL-2 with pentameric OMCP.

FIG. 11A, FIG. 11B and FIG. 11C depict a schematic of the differentialIL2 binding and activation in vivo. (FIG. 11A) Regular wild-type IL2preferentially binds to cells such as CD4⁺Foxp3⁺T_(regs) and vascularendothelium, both of which express the high affinity α chain along withthe signaling 13 and γ chains of the IL2 receptor. (FIG. 11B) The R38Aand F42K mutations in IL2 decrease affinity for the α chain of the IL2receptor. (FIG. 11C) By linking R38A/F42K IL2 to the high affinity NKG2Dligand OMCP delivery and binding of this cytokine to NKG2D-expressingCTLs such as NK cells and activated CD8⁺ T cells is increased. Width ofarrows indicates proposed strength of IL2 binding and/or signaling.

FIG. 12 depicts a schematic of the experimental design of immunotherapyexperiments.

FIG. 13 depicts a schematic of the experimental design of vaccinationexperiments.

FIG. 14A and FIG. 14B depict graphs showing lung cancer susceptible andresistant strains of mice. (FIG. 14A) AJ and 129 mouse strains aresusceptible to lung cancer as evidenced by tumor burden whereas B6 andC3H mouse strains are resistant to lung cancer as evidenced by tumorburden. (FIG. 14B) Upon incubation with freshly isolated NK cells fromthe various mouse strains, B6 and C3H NK cells result in significantlymore LM2 lung carcinoma cell lysis than AJ and 129 NK cells.

FIG. 15 depicts a graph showing that in human men, a greater percentageof NK cells appear to produce TNFα in “resistant” patients versus“susceptible” patients.

FIG. 16 depicts a graph showing that ex vivo cytokine activation canreverse natural killer cell dysfunction. Mouse NK Cells that did notshow significant lysis of cancer cells (NK cells from 129 & AJ strains)were much more effective at lysis when treated with IL-2. NK cells fromcancer-resistant strains also showed increase % of specific lysis.

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E and FIG. 17F depictgraphs showing binding of fluorescently labeled construct tested invitro at 37 degrees in bulk splenocytes. The construct appears to onlybind to NK cells (express NKG2D). Red line is OMCP-IL2 construct. (FIG.17A) DX5+CD3− NK cell; (FIG. 17B) CD4+CD3+ T cells; (FIG. 17C) CD8+ CD3+T cells; (FIG. 17D) CD11C+CD11b− DCs; (FIG. 17E) CD11c−CD11 b+ Macs;(FIG. 17F) CD19+CD3− B cells.

FIG. 18 depicts a schematic dosing regimen for IL2 or IL2 constructs.

FIG. 19 depicts a schematic dosing regimen for IL2 or IL2 constructsafter irradiation.

FIG. 20A, FIG. 20B and FIG. 20C depict images and alignments of the OMCPstructure. (FIG. 20A) Ribbon diagram of CPXV OMCP. Secondary structureelements are noted, S for beta strands and H for helix. The α1/α2portions of the platform domain are indicated in cyan and magenta,respectively. (FIG. 20B) Ribbon diagram of the α1/α2 domain of MICA (PDBidentifier 1HYR), with the α3 domain removed for clarity. Residues thatcontact NKG2D are colored yellow. (FIG. 20C) Structure alignment of OMCPwith NKG2DLs. The mature sequences of OMCP_(BR) (CPXV-BR-018; GenBankaccession number NP_619807; PDB identifier 4FFE) and OMCP_(MPX)(MPXV-ZAR_1979_005-198; N3R; GenBank accession number AAY97396) arealigned with the ectodomain sequences of MICA (1HYR), MICB (1JE6), ULBP3(1KCG), and RAE-1β (1JFM). Known NKG2D contact residues for NKG2DLs areindicated in yellow. Asn residues likely to be glycosylated are noted byblack boxes in panel C and as black side chains in panels A and B.OMCPbr=SEQ ID NO:13; OMCPmpx=SEQ ID NO:14; MICA=SEQ ID NO:15; MICB=SEQID NO:16; ULBP3=SEQ ID NO:17; and RAE-1B=SEQ ID NO:18

FIG. 21 depicts a graph showing OMCP-targeted delivery of IL15. Higherlevels of CD25 are evident when IL15 is delivered by OMCP vs nakedcytokine alone in equimolar doses.

FIG. 22 depicts a graph showing that the D132R mutation in OMCPsignificantly decreases its NKG2D binding. NK expansion and activationin the presence of mutIL2, OMCP-mutIL2, and D132ROMCP-mutIL2 was tested.The D132R mutation ameliorated the superiority of natural killer cellactivation over cytokine alone.

FIG. 23 depicts various embodiments of the invention. 1. depicts OMCPhelix 2 linked to cytokine. 2. depicts pegylation of the composition. 3.depicts a composition comprising engineered glycans. 4. depicts variouslinker lengths and compositions. 5. depicts an antibody linked to acytokine. For example a Fab specific NKG2D antibody. 6. depicts a NKG2DLlinked to a cytokine. For example, MIC or ULBP. 7 depicts an alternativeOMCP linked to a cytokine. For example, OMCP_(mpx) could represent apreferred variant of OMCP, and mutant OMCP could represent either a gainor loss of function for NKG2D binding. 8. depicts re-targeting of theOMCP in a composition. For example, a mutant OMCP may be directed toNKG2A, NKG2C, NKG2E, etc. 9. depicts other viral protein liked to acytokine. For example, the other viral protein may also bind toreceptors on immune cells. 10. depicts OMCP linked to mutant cytokines.It is understood that the OMCP sequence could be from various sourcessuch as cowpox or monkeypox. Also, Fc-chimeras of OMCP and IL2, andvariants thereof may be used.

FIG. 24A and FIG. 24B depict the structure of OMCP in complex withNKG2D. (FIG. 24A) OMCP bound to NKG2D. OMCP is colored magenta and theprotomers of NKG2D are colored cyan (“A”) and yellow (“B”). NKG2DA makescontacts primarily with the H2a helix and NKG2D^(B) with H2b. Mutationsintroduced to facilitate alternate crystal packing are shown in red. TheS193-S194 bond is shown as a ball on each NKG2D protomer. Theasparagines of putative hNKG2D glycosylation sites are shown in orange.The asparagine of the confirmed N-glycan site of OMCP is shown green(data not shown) (FIG. 24B) View of the interface between OMCP-NKG2D.The α2 domain of OMCP is shown in the front with the α1 domain behind.OMCP and NKG2D are shown with cartoon representations for the mainchain, with the side chains of contact residues shown as sticks.Hydrogen bonds and salt bridges are indicated with green dotted lines.

FIG. 25A, FIG. 25B and FIG. 25C depicts the interface of OMCP and NKG2D.(FIG. 25A) The local environment of the OMCP-NKG2D binding interfacesurrounding the D132R residue. The D132R mutation ablates OMCP-NKG2Dbinding. (FIG. 25B) A representative experiment for binding of WT and(D132R) OMCP to NKG2D by SPR. 100 nM of OMCP or (D132R) OMCP wereinjected at 50 μl/min over flowcells containing immobilized biotinylatedmurine NKG2D. (FIG. 25C) Ba/F3 cells transduced with NKG2D, FCRL5, orempty vector were stained with OMCP tetramers (solid line), D132Rtetramers (dashed line), or WNV DIII tetramer control (gray histogram).Representative results from three independent experiments.

FIG. 26A, FIG. 26B, FIG. 26C and FIG. 26D depict the differences in theβ5′-β5 loop (L2) of human and murine NKG2D. (FIG. 26A, FIG. 26B)Superimposition of mNKG2D (grey) (PDB ID: 1HQ8) with the structure ofOMCP-hNKG2D (yellow and cyan). Core binding residues Y152 (Y168) andY199 (Y215) are positionally conserved, while core binding residue M184(I200) is not. (FIG. 26C) Surface representation of OMCP (magenta)interacting with the β5′-β5 loop. (FIG. 26D) Conservation of M184 andQ185. Only the NKG2D of mice, rats, guinea pigs, and flying foxes (notshown) differ. Conservation score is as computed by the ConSurf server.Human, organgutan, chimpanzee, gibbon, macaque-SEQ ID NO:19; Greenmonkey-SEQ ID NO:20; Marmoset-SEQ ID NO:21; Mouse-SEQ ID NO:22; Rat-SEQID NO:23; Guinea pig-SEQ ID NO:24; Ground squirrel-SEQ ID NO:25; Deermouse-SEQ ID NO:26; Naked mole rat-SEQ ID NO:27; Prairie vole-SEQ IDNO:28; European shrew-SEQ ID NO:29; Star-nosed mole-SEQ ID NO:30;Chinese hamster-SEQ ID NO:31; Cat-SEQ ID NO:32.

FIG. 27A, FIG. 27B, FIG. 27C, FIG. 27D, FIG. 27E, FIG. 27F, FIG. 27G,FIG. 27H and FIG. 27I depict a novel NKG2D binding adaptation. Surfacerepresentation of NKG2D and surface and cartoon representations of OMCP,MICA and ULBP3. Buried surface areas for NKG2DA and NKG2D^(B) areindicated in cyan and yellow, respectively. Buried surface area by NKG2Dis indicated for OMCP (magenta), MICA (green), and ULBP3 (orange). Thecore binding residues of NKG2D and NKG2D-binding elements of NKG2DLs areindicated. NKG2D (FIG. 27A) and OMCP (FIG. 27B, FIG. 27C) bindinginteractions. NKG2D (FIG. 27D) and MICA (FIG. 27E, FIG. 27F) bindinginteractions. NKG2D (FIG. 27G) and ULBP3 (FIG. 27H, FIG. 27I) bindinginteractions. (FIG. 27J) Alignment by secondary structure of NKG2DLs(PDB ID: OMCP (4FFE), MICA (1HYR), MICB (1JE6), ULBP3 (1KCG) and RAE-1β(1JSK)). Contact residues are indicated for OMCP (magenta), MICA(green), ULBP3 (orange) and RAE-1β (bold and italics). Secondarystructure elements are noted above the sequence (arrow for beta sheets,cylinders for alpha helices). Predicted glycan sites are highlighted inblack. OMCPbr=SEQ ID NO:13; OMCPmpx=SEQ ID NO:14; MICA=SEQ ID NO:15;MICB=SEQ ID NO:16; ULBP3=SEQ ID NO:17; and RAE-1B=SEQ ID NO:18

FIG. 28A, FIG. 28B, FIG. 28C, FIG. 28D and FIG. 28E depict activation ofNK cells by cell-associated OMCP. Model depicting NKG2D interaction with(FIG. 28A) host, (FIG. 28B) cancer-induced, (FIG. 28C) viral, or (FIG.28D) chimeric ligands. Binding interactions that lead to NKG2D-mediatedsignaling are indicated by DAP10 tyrosine phosphorylation (red filledcircles). (FIG. 28E) IL-2-activated splenocytes were used as cytotoxiceffectors against stably transduced Ba/F3 cell lines. Splenocytes wereactivated with 200 U/ml of IL-2 for 24 hours. Labeled target cells wereco-incubated with activated splenocytes for 4 hours at effector:targetratios of 10:1, 20:1, and 40:1. Killing was measured by incorporation of7AAD by CFSE-labeled target cells using flow cytometry. Representativedata from five independent experiments is shown

FIG. 29A and FIG. 29B depict the electron density supporting a cispeptide conformation. Stereo view of the β5-β6 loop of hNKG2D. Residues193-Ala-Ser-Ser-Phe-Lys-197 (SEQ ID NO:33) is displayed for theOMCP-hNKG2D structure (yellow) and the structure of hNKG2D alone (grey).The 2Fo-Fc map for OMCP-hNKG2D is displayed at 2a.

FIG. 30A and FIG. 30B depicts graphs showing survival curves of C57Bl/6Jmice following infection with West Nile Virus (WNV). Mice were treatedwith OMCP-IL2, OMCP(D132R)-IL2, IL2, IL(38R/42A) or PBS after infectionwith WNV. Infection with OMCP-IL2 and IL2(38R/42A) resulted in survivalbeyond 21 days in 40% of mice compared to 0 mice following treatmentwith PBS or OMCP(D132R)-IL2.

FIG. 31A, FIG. 31B, FIG. 31C and FIG. 31D depicts flow cytometry datashowing that OMCP-Mutant IL2 activates NK and CD8+ T cells. FIG. 31Ashows that a relatively higher proportion of NK cells was evident in theOMCP-mutant IL2 group. FIG. 31B shows that perforin levels were higherin OMCP-mutant IL2 treated NK cells (red) compared to saline (black),IL2 (blue) or mutant IL2 (green) treated ones. FIG. 31C shows thatsimilar to NK cells, higher intracellular levels of perforin wereevident in CD8+ T cells treated with OMCP-mutant IL2 compared to otherconditions. FIG. 31D shows that when gating on CD4+Foxp3+CD45RA− T cellsa relatively higher proportion of activated CD25+CD127− regulatory Tcells was evident in IL2 treated peripheral blood lymphocyte culturescompared to other conditions.

FIG. 32 depicts a schematic of the various IL18-OMCP constructs. Threeversions were made, each having OMCP attached to either WT human IL-18,WT murine IL-18, or mutant human IL-18 (which inhibits its interactionwith IL-18BP).

FIG. 33 depicts a flow cytometry plot showing that IL18-OMCP activatesNK cells. Peripheral blood lymphocytes were cultured for 48 hours in 4.4μM of either wild-type IL18 (blue), OMCP-IL18 (red) or saline (black).Activation of CD56+CD3-Natural killer cells, as measured by surface CD69expression, was superior by OMCP-IL18 compared to wild-type IL18.

DETAILED DESCRIPTION OF THE INVENTION

The compositions and methods described herein provide for delivery ofcytokines to a defined cell via a ligand. The fusion of a cytokine to aligand which specifically binds to a protein on the target cell createsan “address” for delivery of the cytokine. Specifically, using theinvention disclosed herein, IL2 is directly targeted to lymphocytes,such as natural killer (NK) cells and CD8+ cytotoxic T lymphocytes(CTLs), via the orthopoxvirus major histocompatibility complex classI-like protein (OMCP) ligand. However other NKG2D ligands, including butnot limited to ULBP1, ULBP2, ULBP3, H60, Rae-1α, Rae-1β, Rae-1δ, Rae-1γ,MICA, MICB, h-HLA-A, could also be used instead of OMCP. Specificdelivery of IL2 to lymphocytes will enhance the efficacy of IL2, whichcould lead to reduced dosages and a significant decrease in associatedtoxicity. This methodology may be used for other cytokines, including,but not limited to, IL15, IL18 and interferons.

Specific aspects of the invention are described in detail below.

I. Composition

In an aspect, the invention encompasses a composition comprising acytokine linked to a ligand. The composition may further comprise alinker to connect the cytokine to the ligand. The cytokine, ligand andlinker are described in greater detail below. It should be understoodthat any of the cytokines described in detail below can be linked to anyof the ligands described in detail below in the absence or presence ofany of the linkers described below. In another aspect, the inventionprovides a nucleic acid molecule encoding a cytokine, a ligand, andoptionally a linker.

(a) Cytokine

As used herein, a “cytokine” is a small protein (˜5-20 kDa) that isimportant in cell signaling. Cytokines are released by cells and affectthe behavior of other cells and/or the cells that release the cytokine.Non-limiting examples of cytokines include chemokines, interferons,interleukins, lymphokines, tumor necrosis factor, monokines, and colonystimulating factors. Cytokines may be produced by a broad range of cellsincluding, but not limited to, immune cells such as macrophages, Blymphocytes, T lymphocytes, mast cells and monocytes, endothelial cells,fibroblasts and stromal cells. A cytokine may be produced by more thanone type of cell. Cytokines act through receptors and are especiallyimportant in the immune system, modulate the balance between humoral andcell-based immune responses, and regulate maturation, growth andresponsiveness of cell populations. Cytokines are important in hostresponses to infection, immune responses, inflammation, trauma, sepsis,cancer and reproduction. A cytokine of the invention may be a naturallyoccurring cytokine or may be a mutated version of a naturally occurringcytokine. As used herein, “naturally occurring”, which may also bereferred to as wild-type, includes allelic variances. A mutated versionor “mutant” of a naturally occurring cytokine refers to specificmutations that have been made to the naturally occurring sequence toalter the function, activity and/or specificity of the cytokine. In oneembodiment, the mutations may enhance the function, activity and/orspecificity of the cytokine. In another embodiment, the mutations maydecrease the function, activity and/or specificity of the cytokine. Themutation may include deletions or additions of one or more amino acidresidues of the cytokine.

Cytokines may be classified based on structure. For example, cytokinesmay be classified into four types: the four-α-helix bundle family, theIL1 family, the IL17 family and the cysteine-knot cytokines. Members ofthe four-α-helix bundle family have three-dimensional structures withfour bundles of α-helices. This family is further divided into threesub-families: the IL2 subfamily, the interferon (IFN) subfamily and theIL10 subfamily. The IL2 subfamily is the largest and comprises severalnon-immunological cytokines including, but not limited to,erythropoietin (EPO) and thrombopoietin (TPO). In certain embodiments, acytokine of the composition is a cytokine from the four-α-helix bundlefamily or a mutant thereof. A skilled artisan would be able to determinecytokines within the four-α-helix bundle family. In other embodiments, acytokine of the composition is an IL2 subfamily cytokine or a mutantthereof. Non-limiting examples of members of the IL2 subfamily includeIL2, IL4, IL7, IL9, IL15 and IL21. In a specific embodiment, a cytokineof the composition is IL2 or a mutant thereof. In certain embodiments, acytokine of the composition is IL15 or a mutant thereof. The sequenceinformation for the full length human IL15 amino acid sequence can befound using, for example, the GenBank accession number CAG46777.1,AAI00962.1 or AAI00963.1. The sequence information for the full lengthhuman 11_15 mRNA sequence can be found using, for example, the GenBankaccession number CR542007.1, KJ891469.1, NM_172175.2, NM_000585.4 orCR541980.1. A skilled artisan will appreciate that IL15 may be found ina variety of species and methods of identifying analogs or homologs ofIL15 are known in the art as described in detail below.

In another embodiment, a cytokine of the invention is an IL1 familycytokine or a mutant thereof. The IL1 family is a group of 11 cytokines,which plays a central role in the regulation of immune and inflammatoryresponses. Generally, the IL1 family of cytokines are proinflammatorycytokines that regulate and initiate inflammatory responses.Non-limiting examples of IL1 family cytokines include IL1α, IL1β, IL1Ra,IL18, IL36Ra, IL36α, IL37, IL36β, IL36γ, IL38, and IL33. IL1 familymembers have a similar gene structure. A skilled artisan would be ableto determine cytokines within the IL1 family. In certain embodiments, acytokine of the composition is IL18 or a mutant thereof. The sequenceinformation for the full length human IL18 amino acid sequence can befound using, for example, the GenBank accession number CAG46771.1. Thesequence information for the full length human IL18 mRNA sequence can befound using, for example, the GenBank accession number KR710147.1,CR542001.1, CR541973.1 or KJ1897054.1. A skilled artisan will appreciatethat IL18 may be found in a variety of species and methods ofidentifying analogs or homologs of IL18 are known in the art asdescribed in detail below.

In other embodiments, a cytokine of the composition is an interferonsubfamily cytokine or a mutant thereof. Interferons are named for theirability to “interfere” with viral replication by protecting cells fromvirus infection. IFNs also have other functions: they activate immunecells, such as natural killer cells and macrophages; they increase hostdefenses by up-regulating antigen presentation by virtue of increasingthe expression of major histocompatibility complex (MHC) antigens. Basedon the type of receptor through which they signal, human interferonshave been classified into three major types: Type I IFN, Type II IFN,and Type III IFN. Type I IFNs bind to a specific cell surface receptorcomplex known as the IFN-α/β receptor (IFNAR) that consists of IFNAR1and IFNAR2 chains. Non-limiting examples of type I interferons presentin humans are IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω. Thus, in certainembodiments, a cytokine of the composition is a Type 1 IFN cytokine or amutant thereof, including, but not limited to wild-type and mutant formsof IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω. Type II IFNs bind to IFNGR thatconsists of IFNGR1 and IFNGR2 chains. Non-limiting examples of type IIinterferons present in humans is IFN-γ. Thus, in certain embodiments, acytokine of the composition is a Type II IFN cytokine or a mutantthereof, including, but not limited to wild-type and mutant forms ofIFN-γ. Type III IFNs signal through a receptor complex consisting ofIL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12).Non-limiting examples of type III interferons include IFN-λ1, IFN-λ2 andIFN-λ3 (also called IL29, IL28A and IL28B respectively). Thus, incertain embodiments, a cytokine of the composition is a Type III IFNcytokine or a mutant thereof, including, but not limited to wild-typeand mutant forms of IFN-A1, IFN-A2 and IFN-A3.

In certain embodiments, a cytokine of the invention is an interleukin ormutant thereof. The majority of interleukins are synthesized by helperCD4 T lymphocytes, as well as through monocytes, macrophages, andendothelial cells. Interleukins may promote the development anddifferentiation of T and B lymphocytes and hematopoietic cells.Non-limiting examples of interleukins include IL1, IL2, IL3, IL4, IL5,IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16,IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28,IL29, IL30, IL31, IL32, IL33, IL35, or IL36. Thus, in certainembodiments, a cytokine of the composition is an interleukin or a mutantthereof, including, but not limited to wild-type and mutant forms ofIL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12,IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24,IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL36. Ina specific embodiment, a cytokine of the composition is IL2 or a mutantthereof. IL2 is a lymphokine that induces the proliferation ofresponsive T cells. In addition, it acts on some B cells, viareceptor-specific binding, as a growth factor and antibody productionstimulant. The IL2 protein is secreted as a single glycosylatedpolypeptide, and cleavage of a signal sequence is required for itsactivity. The structure of IL2 comprises a bundle of 4 helices (termedA-D), flanked by 2 shorter helices and several poorly defined loops.Residues in helix A, and in the loop region between helices A and B, areimportant for receptor binding. Secondary structure analysis suggestssimilarity to IL4 and granulocyte-macrophage colony stimulating factor(GMCSF). In a specific embodiment, a cytokine of the composition is IL2or a variant thereof. A variant may be a truncated or mutated IL2. Thesequence information for the full length human IL2 amino acid sequencecan be found using, for example, the GenBank accession number AAA59140.1or AAH70338.1. The sequence information for the full length human IL2mRNA sequence can be found using, for example, the GenBank accessionnumber BC070338.1 or M22005.1.

A skilled artisan will appreciate that IL2 may be found in a variety ofspecies. Non-limiting examples include mouse (AAI16874.1), pig(NP_999026.1), cattle (AAQ10670.1), rat (EDM01295.1), rabbit(AAC23838.1), goat (AAQ10671.1), sheep (ABK41601.1), chicken(AAV35056.1), hamster (ERE88380.1), and dog (AAA68969.1). It isappreciated that the present invention is directed to analogs of IL2 inother organisms and is not limited to the human analog. Homologs can befound in other species by methods known in the art. For example,sequence similarity may be determined by conventional algorithms, whichtypically allow introduction of a small number of gaps in order toachieve the best fit. In particular, “percent identity” of twopolypeptides or two nucleic acid sequences is determined using thealgorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA87:2264-2268, 1993). Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (J. Mol. Biol. 215:403-410,1990). BLAST nucleotide searches may be performed with the BLASTNprogram to obtain nucleotide sequences homologous to a nucleic acidmolecule of the invention. Equally, BLAST protein searches may beperformed with the BLASTX program to obtain amino acid sequences thatare homologous to a polypeptide of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., BLASTX and BLASTN) are employed. Seewww.ncbi.nlm.nih.gov for more details. Generally a homolog will have aleast 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89% homology. In anotherembodiment, the sequence may be at least 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% homologous to IL2.

In a specific embodiment, a cytokine of the composition is a wildtypesequence of IL2 such as the sequence set forth in SEQ ID NO:5(APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT). In an alternative embodiment, a cytokine is a mutatedversion of IL2. In an embodiment, a mutation is a mutation that causesIL2 to preferentially bind the receptor IL2βγ. In another embodiment, amutation is a mutation that alters the function of IL2 such that IL2 hasa decreased affinity for the IL2 receptor alpha (IL2Rα). For example, amutation may be one or more mutations selected from the group consistingof R38A, F42K and/or C125S relative to SEQ ID NO:5. The C125S mutationmay be included to reduce protein aggregation. In a specific embodiment,a mutated version of IL2 comprises at least one mutation selected fromthe group consisting of R38A, F42K and C125S relative to SEQ ID NO:5. Inanother specific embodiment, a mutated version of IL2 comprises themutations R38A, F42K and C125S relative to SEQ ID NO:5. In a specificembodiment, a cytokine of the composition is a mutated sequence of IL2such as the sequence set forth in SEQ ID NO:6(APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT).

In an alternative aspect, a toxin is substituted for a cytokine. Theterm “toxin” means the toxic material or product of plants, animals,microorganisms (including, but not limited to, bacteria, viruses, fungi,rickettsiae or protozoa), or infectious substances, or a recombinant orsynthesized molecule, whatever their origin and method of production. Atoxin may be a small molecule, peptide, or protein that is capable ofcausing disease on contact with or absorption by body tissuesinteracting with biological macromolecules such as enzymes or cellularreceptors. A toxin may be a “biotoxin” which is used to explicitlyidentify the toxin as from biological origin. Biotoxins may be furtherclassified into fungal biotoxins, or short mycotoxins, microbialbiotoxins, plant biotoxins, short phytotoxins and animal biotoxins.Non-limiting examples of biotoxins include: cyanotoxins, produced bycyanobacteria, such as microcystins, nodularins, anatoxin-a,cylindrospermopsins, lyngbyatoxin-a, saxitoxin, lipopolysaccharides,aplysiatoxins, BMAA; dinotoxins, produced by dinoflagellates, such assaxitoxins and gonyautoxins; necrotoxins produced by, for example, thebrown recluse or “fiddle back” spider, most rattlesnakes and vipers, thepuff adder, Streptococcus pyogenes; neurotoxins, produced by, forexample, the black widow spider, most scorpions, the box jellyfish,elapid snakes, the cone snail, the Blue-ringed octopus, venomous fish,frogs, palythoa coral, various different types of algae, cyanobacteriaand dinoflagellates, such as botulinum toxin (e.g. Botox), tetanustoxin, tetrodotoxin, chlorotoxin, conotoxin, anatoxin-a, bungarotoxin,caramboxin, curare; myotoxins, found in, for example, snake and lizardvenoms; and cytotoxins such as ricin, from castor beans, apitoxin, fromhoney bees, and T-2 mycotoxin, from certain toxic mushrooms. In certainembodiments, a toxin is a cytotoxin. In an embodiment, a cytotoxin isselected from the group consisting of ricin, apitoxin, and T-2mycotoxin. In a specific embodiment, a toxin is ricin.

In certain embodiments, a cytokine or toxin of the invention may bePEGylated for improved systemic half-life and reduced dosage frequency.In an embodiment, PEG may be added to a cytokine or toxin. As such, acomposition of the invention may comprise a cytokine or toxin comprisingPEG. In an embodiment, PEG may be selected from the group consisting ofPEG-10K, PEG-20K and PEG-40K. Methods of conjugating PEG to a proteinare standard in the art. For example, see Kolate et al, Journal ofControlled Release 2014; 192(28): 67-81, which is hereby incorporated byreference in its entirety. Still further, a cytokine or toxin of theinvention may be modified to remove T cell epitopes. T cell epitopes canbe the cause of an immunogenicity issue upon administration of acomposition to a subject. Through their presentation to T cells, theyactivate the process of anti-drug antibody development. Preclinicalscreening for T cell epitopes may be performed in silico, followed by invitro and in vivo validation. T cell epitope-mapping tools such asEpiMatrix can be highly accurate predictors of immune response.Deliberate removal of T cell epitopes may reduce immunogenicity. Othermeans of improving the safety and efficacy of a composition of theinvention by reducing their immunogenicity include humanization andPEGylation.

(b) Ligand

As used herein, a “ligand” is a protein that specifically binds to areceptor on a target cell and is not the corresponding binding partnerto the cytokine linked to the ligand. A ligand may be from a eukaryote,a prokaryote or a virus. In certain embodiments, a ligand may be from avirus. The phrase “specifically binds” herein means ligands bind to thetarget protein with an affinity (K_(d)) in the range of at least 0.1 mMto 1 μM, or in the range of at least 0.1 μM to 200 nM, or in the rangeof at least 0.1 μM to 10 nM. A dissociation constant (K_(d)) measuresthe propensity of a larger object to separate (dissociate) reversiblyinto smaller components. The dissociation constant is the inverse of theassociation constant. The dissociation constant may be used to describethe affinity between a ligand (L) and a target protein (P). As such,K_(d)=([P]×[L])/[C], wherein C is a ligand-target protein complex andwherein [P], [L] and [C] represent molar concentrations of the protein,ligand and complex, respectively. Methods of determining whether aligand binds to a target protein are known in the art. For instance, seethe Rossi and Taylor, Nature Protocols 2011; 6: 365-387.

A ligand may trigger a signal through its binding to a receptor on atarget cell. A receptor is a protein molecule that may be embeddedwithin the plasma membrane surface of a cell that receives chemicalsignals from outside the cell. When such chemical signals bind to areceptor, they cause some form of cellular/tissue response. In preferredembodiments, a target cell is an immune cell. Accordingly, a ligand ofthe composition binds to a receptor expressed on immune cells.Non-limiting example of immune cells include macrophages, B lymphocytes,T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils,natural killer cells, basophils, neutrophils. Thus, in certainembodiments, immune cells include, but are not limited to, macrophages,B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells,eosinophils, natural killer cells, basophils, neutrophils. In a specificembodiment, an immune cell is a natural killer cell or a T lymphocyte.Non-limiting examples of receptors expressed on immune cells includemajor histocompatibility complex (MHC; e.g. MHCI, MHCII, and MHCIII),toll-like receptors (TLRs; e.g. TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13), CD94/NKG2 familyreceptor, endothelin receptors, signaling lymphocytic activationmolecule (SLAM) family of receptors. Thus, in certain embodiments, areceptor on a target cell includes, but is not limited to, majorhistocompatibility complex (MHC; e.g. MHCI, MHCII, and MHCIII),toll-like receptors (TLRs; e.g. TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13), CD94/NKG2 familyreceptor, endothelin receptors, signaling lymphocytic activationmolecule (SLAM) family of receptors. In a specific embodiment, thereceptor on a target cell is a CD94/NKG2 family receptor. In anotherspecific embodiment, a ligand of the composition specifically binds to areceptor expressed on natural killer (NK) cells and CD8+ cytotoxic Tlymphocytes (CTLs). In preferred embodiments, a ligand of thecomposition does not specifically bind to a receptor on vascularendothelial cells or regulatory T cells (T_(regs)).

A receptor expressed on NK cells and CTLs may be a CD94/NKG2 familyreceptor or KLRG1. KLRG1 (Killer cell lectin-like receptor subfamily Gmember 1) is a protein that in humans is encoded by the KLRG1 gene.CD94/NKG2 family receptors are a family of C-type lectin receptors whichare expressed predominantly on the surface of NK cells and a subset ofCD8+T-lymphocyte. These receptors stimulate or inhibit cytotoxicactivity of NK cells, therefore they are divided into activating andinhibitory receptors according to their function. CD94/NKG2 recognizeMHC class I--related glycoproteins. CD94/NKG2 family includes sevenmembers: NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F and NKG2H. Thus, incertain embodiments, a ligand of the invention specifically binds to areceptor selected from the group consisting of NKG2A, NKG2B, NKG2C,NKG2D, NKG2E, NKG2F and NKG2H. NKG2 receptors are transmembrane proteinstype II which dimerize with CD94 molecule. CD94 contains a shortcytoplasmic domain and it is responsible for signal transduction.Therefore NKG2 receptors form disulfide bonded heterodimers. NKG2Drepresents an exception, it is a homodimer. NKG2A and NKG2B receptorstransmit inhibitory signal. NKG2C, NKG2E and NKG2H are activatingreceptors. NKG2D is activating receptor as well but it couples withadaptor protein DAP10 which carries signaling motif YINM (SEQ ID NO:34).Src or Jak kinases phosphorylate DAP10, which can then associate withp85 subunit of PI(3)K or adaptor molecule Grb2. This signaling triggersactin reorganization (cell polarization) and degranulation. NKG2Freceptor function has not been clarified yet.

In a specific embodiment, a ligand of the composition specifically bindsto the NKG2D receptor. NKG2D is an activating receptor found on NK cellsand CD8+ T cells (both αβ and γδ). The structure of NKG2D consists oftwo disulphide-linked type H transmembrane proteins with shortintracellular domains incapable of transducing signals. The function ofNKG2D on CD8+ T cells is to send co-stimulatory signals to activatethem. In an embodiment, a ligand that binds to NKG2D may be ananti-NKG2D antibody. An “anti-NKG2D” includes all antibodies thatspecifically bind an epitope within NKG2D. The term “antibody’ includesthe term “monoclonal antibody”. “Monoclonal antibody” refers to anantibody that is derived from a single copy or clone, including e.g.,any eukaryotic, prokaryotic, or phage clone. Monoclonal antibodies canbe produced using e.g., hybridoma techniques well known in the art, aswell as recombinant technologies, phage display technologies, synthetictechnologies or combinations of such technologies and other technologiesreadily known in the art. Further by “antibody” is meant a functionalmonoclonal antibody, or an immunologically effective fragment thereof;such as an Fab, Fab′, or F(ab′)2 fragment thereof. As long as theprotein retains the ability specifically to bind its intended target, itis included within the term “antibody.” Also included within thedefinition “antibody” for example are single chain forms, generallydesignated Fv, regions, of antibodies with this specificity. These scFvsare comprised of the heavy and light chain variable regions connected bya linker. Methods of making and using scFvs are known in the art.Additionally, included within the definition “antibody” aresingle-domain antibodies, generally designated sdAb, which is anantibody fragment consisting of a single monomeric variable antibodydomain. A sdAb antibody may be derived from camelids (V_(H)H fragments)or cartilaginous fishes (V_(NAR) fragments). As used herein “humanizedantibody” includes an anti-NKG2D antibody that is composed partially orfully of amino acid sequence sequences derived from a human antibodygerm line by altering the sequence of an antibody having non-humancomplementarity determining regions (“CDR”). The simplest suchalteration may consist simply of substituting the constant region of ahuman antibody for the murine constant region, thus resulting in ahuman/murine chimera which may have sufficiently low immunogenicity tobe acceptable for pharmaceutical use. Preferably, however, the variableregion of the antibody and even the CDR is also humanized by techniquesthat are by now well known in the art. The framework regions of thevariable regions are substituted by the corresponding human frameworkregions leaving the non-human CDR substantially intact, or evenreplacing the CDR with sequences derived from a human genome. CDRs mayalso be randomly mutated such that binding activity and affinity forNKG2D is maintained or enhanced in the context of fully human germlineframework regions or framework regions that are substantially human. Incertain embodiments, an anti-NKG2D antibody is a Fab, Fab′, or F(ab′)₂fragment.

In another embodiment, ligands that bind to NKG2D share an MHC classI-related α1α2 superdomain that constitutes the common site forinteraction with NKG2D. Non-limiting examples of ligands that bind toNKG2D include MHC class I-related glycoproteins such as MIC familyproteins (i.e., MICA, MICB), UL16-binding family proteins (i.e., ULBP1,ULBP2, ULPB3, ULBP4, ULBP5, ULBP6), retinoid acid early induce gene 1(Rae1)-like proteins (i.e., Rae1α, Rae1β, Rae1γ, Rae1δ, Rae1ε), membersof the H60 protein family (i.e., H60a, H60b, H60c), h-HLA-A, as well asMult1 in mice and orthopoxvirus major histocompatibility complex classI-like protein (OMCP). In certain embodiments, a ligand is a MHCclass-I-related glycoprotein. In other embodiments, a ligand of theinvention is selected from the group consisting of MICA, MICB, ULBP1,ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae1α, Rae1β, Rae1γ, Rae1δ, Rae1ε,H60a, H60b, H60c, h-HLA-A, Mult1 and OMCP. In an embodiment, a ligand isa UL16-binding family protein or a MIC family protein. In a specificembodiment, a ligand is selected from the group consisting of ULBP1,ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In another specific embodiment, aligand is ULBP3. In a specific embodiment, a ligand is orthopoxvirusmajor histocompatibility complex class I-like protein (OMCP) or avariant thereof. A variant may be a truncated or mutated OMCP that hasabout the same binding affinity of the full length OMCP. In anembodiment, a variant may be a truncated or mutated OMCP that has aslightly lower binding affinity relative to the binding affinity of thefull length OMCP. In another embodiment, a variant is a truncated ormutated OMCP that has a slightly higher binding affinity relative to thebinding affinity of the full length OMCP. Methods to determine bindingaffinity of a ligand to target protein are known in the art anddescribed above. OMCP specifically binds to NKG2D with a bindingaffinity of about 0.1 to about 5 nM. For example, OMCP specially bindsto human NKG2D with a binding affinity of about 0.2 nM and mouse NKG2Dwith a binding affinity of about 3 nM. In a preferred embodiment, OMCPor a variant thereof binds to human NKG2D with a binding affinity ofabout 1000 nM to about 0.1 nM. In certain embodiments, OMCP or a variantthereof binds to human NKG2D with a binding affinity of about 100 nM toabout 0.1 nM, about 10 nM to about 0.1 nM, or about 1 nM to about 0.1nM. In other embodiments, OMCP or a variant thereof binds to human NKG2Dwith a binding affinity of about 1000 nM to about 1 nM, or about 1000 nMto about 10 nM, or about 1000 nM to about 100 nM. In still otherembodiments, OMCP or a variant thereof binds to human NKG2D with abinding affinity of about 100 nM to about 1 nM, or about 100 nM to 10nM. For example, OMCP or a variant thereof binds to human NKG2D with abinding affinity of about 1000 nM, about 500 nM, about 100 nM, about 50nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM about 5nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about0.3 nM, about 0.2 nM or about 0.1 nM. In another embodiment, a variantis a truncated or mutated OMCP that has binding affinity for one or moreNKG2 family receptors other than NKG2D. For example, a variant is atruncated or mutated OMCP that has binding affinity for one or more NKG2family receptors selected from the group consisting of NKG2A, NKG2B,NKG2C, NKG2E, NKG2F and NKG2H. Mutations to OMCP may be rationallyselected via structure-based knowledge or mutations to OMCP may beidentified via selection-based mutagenesis. In certain embodiments,mutations may be rationally selected to occur in the OMCP-NKG2Dinterface to either enhance or reduce binding affinity. Amino acidsinvolved in binding at the OMCP-NKG2D interface are described in theExamples.

The structure of OMCP consists of an MHCI-like α1/α2 platform domain(FIG. 20A). The platform domain of OMCP has been trimmed to have only asix-stranded beta sheet with shorter flanking helices. The helix of theOMCP α1 domain (H1) is continuous, while the helix of the α2 domain isbroken into two regions (H2a and H2b). The helices flank a six-strandedbeta sheet and together form the characteristic platform that definesMHC proteins. Like other NKG2DLs (FIG. 20B), the alpha helices of OMCPare close together and thus have no groove for binding peptides or otherligands like antigen-presenting MHC platform domains. OMCP contains onedisulfide bond between S5 and H2b, and this disulfide bond is conservedin most NKG2DLs (FIG. 20C). In certain embodiments, a ligand of theinvention comprises one or more of the α helices of a MHC classI-related glycoprotein. In other embodiments, a ligand of the inventionconsists of one or more of the α helices of a MHC class I-relatedglycoprotein. More specifically, a ligand of the invention comprises theα1 domain (H1), α2 domain (H2), H2a, H2b, or combinations thereof of aMHC class I-related glycoprotein. Or, a ligand of the invention consistsof the α1 domain (H1), α2 domain (H2), H2a, H2b, or combinations thereofof a MHC class I-related glycoprotein. In a specific embodiment, aligand of the invention comprises the α2 domain (H2) of a MHC classI-related glycoprotein. In another specific embodiment, a ligand of theinvention consists of the α2 domain (H2) of a MHC class I-relatedglycoprotein. A skilled artisan would be able to determine the locationof the α helices in other MHC class I-related glycoproteins, forexample, using sequence alignment (see FIG. 20C, which is reproducedfrom Lazear et al. J Virol 2013; 87(2): 840-850, which is herebyincorporated by reference in its entirety). In an embodiment, a ligandof the invention comprises one or more of the α helices of OMCP. Inanother embodiment, a ligand of the invention comprises the α1 domain(H1), α2 domain (H2), H2a, H2b, or combinations thereof of OMCP. Instill another embodiment, a ligand of the invention comprises the α2domain (H2) of OMCP. In a specific embodiment, a ligand of the inventionconsists of one or more of the α helices of OMCP. In another specificembodiment, a ligand of the invention consists of the α1 domain (H1), α2domain (H2), H2a, H2b, or combinations thereof of OMCP. In still anotherspecific embodiment, a ligand of the invention consists of the α2 domain(H2) of OMCP.

The sequence information for the full length OMCP amino acid sequencecan be found using, for example, the GenBank accession number 4FFE_Z,4FFE_Y or 4FFE_X. A skilled artisan will appreciate that homologs ofOMCP may be found in other species or viruses. For example, seeLefkowitz et al, Nucleic Acids Res 2005; 33: D311-316, which is hereinincorporated by reference in its entirety, which describes eighteen OMCPvariants between cowpox and monkeypox virus strains. In an embodiment,OMCP is from an orthopoxvirus. In a specific embodiment, OMCP is from acowpox virus or a monkeypox virus. In another specific embodiment, OMCPis from the Brighton Red strain of cowpoxvirus. Homologs can be found inother species by methods known in the art. For example, sequencesimilarity may be determined by conventional algorithms, which typicallyallow introduction of a small number of gaps in order to achieve thebest fit. In particular, “percent identity” of two polypeptides or twonucleic acid sequences is determined using the algorithm of Karlin andAltschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1993). Such analgorithm is incorporated into the BLASTN and BLASTX programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches may be performed with the BLASTN program to obtain nucleotidesequences homologous to a nucleic acid molecule of the invention.Equally, BLAST protein searches may be performed with the BLASTX programto obtain amino acid sequences that are homologous to a polypeptide ofthe invention. To obtain gapped alignments for comparison purposes,Gapped BLAST is utilized as described in Altschul et al. (Nucleic AcidsRes. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) are employed. See www.ncbi.nlm.nih.gov for moredetails. Generally a homolog will have a least 80, 81, 82, 83, 84, 85,86, 87, 88, or 89% homology. In another embodiment, the sequence may beat least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous toOMCP.

A skilled artisan will appreciate that structural homologs of OMCP maybe found in other species or viruses. A structural homolog may be aprotein that is structurally related but the sequence is a distalhomolog. For example, OMCP has low sequence identity for endogenousNKG2D ligands however it was discovered that OMCP would bind to NKG2Dbased on structural homology. Structural homologs can be found in otherspecies by methods known in the art. For example, protein structureprediction may be determined by various databases, such as Phyre andPhyre2. Such databases generate reliable protein models that may be usedto determine structural homologs. The main results table in Phyre2provides confidence estimates, images and links to the three-dimensionalpredicted models and information derived from either StructuralClassification of Proteins database (SCOP) or the Protein Data Bank(PDB) depending on the source of the detected template. For each match alink takes the user to a detailed view of the alignment between the usersequence and the sequence of known three-dimensional structure. Seewww.sbg.bio.ic.ac.uk/phyre2/ for more details. Generally, a structuralhomolog will have a least 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59%confidence with OMCP. In an embodiment, a structural homolog will have aleast 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69% confidence with OMCP.In another embodiment, a structural homolog will have a least 70, 71,72, 73, 74, 75, 76, 77, 78, or 79% confidence with OMCP. In stillanother embodiment, a structural homolog will have a least 80, 81, 82,83, 64, 85, 86, 87, 88, or 89% confidence with OMCP. In still yetanother embodiment, a structural homolog may have at least 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100% confidence with OMCP. The structuralinformation for OMCP-human NKG2D may be found using the PDB ID: 4PDC.

In a specific embodiment, a ligand of the composition is a sequence ofOMCP such as the sequence set forth in SEQ ID NO:7(HKLAFNFNLEINGSDTHSTVDVYLDDSQIITFDGKDIRPTIPFMIGDEIFLPFYKNVFSEFFSLFRRVPTSTPYEDLTYFYECDYTDNKSTFDQFYLYNGEEYTVKTQEATNKNMWLTTSEFRLKKWFDGEDCIMHLRSLVRKMEDSKRNT). In an embodiment, a ligand of thecomposition is a sequence of OMCP comprising at least 80% identity toSEQ ID NO:7.

For example, the ligand may have about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ IDNO:7.

In another specific embodiment, a ligand of the composition is asequence of OMCP such as the sequence set forth in SEQ ID NO:13(GHKLAFNFNLEINGSDTHSTVDVYLDDSQIITFDGKDIRPTIPFMIGDEIFLPFYKNVFSEFFSLFRRVPTSTPYEDLTYFYECDYTDNKSTFDQFYLYNGEEYTVKTQEATNKNMWLTTSEFRLKKWFDGEDCIMHLRSLVRKMEDSKR). In an embodiment, a ligand of thecomposition is a sequence of OMCP comprising at least 80% identity toSEQ ID NO:13. For example, the ligand may have about 80%, about 81%,about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%identity to SEQ ID NO:13.

In still another specific embodiment, a ligand of the composition is asequence of OMCP such as the sequence set forth in SEQ ID NO:14(HKLVHYFNLKINGSDITNTADILLDNYPIMTFDGKDIYPSIAFMVGNKLFLDLYKNIFVEFFRLFRVSVSSQYEELEYYYSCDYTNNRPTIKQHYFYNGEEYTEIDRSKKATNKNSWLITSGFRLQKWFDSEDCIIYLRSLVRRMEDSNK). In an embodiment, a ligand of thecomposition is a sequence of OMCP comprising at least 80% identity toSEQ ID NO:14.

For example, the ligand may have about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ IDNO:14.

In an alternative aspect, a receptor expressed on immune cells may bePD1. PD1, also known as programmed cell death protein 1 and CD279(cluster of differentiation 279), is a protein that in humans is encodedby the PDCD1 gene. PD1 is a cell surface receptor that belongs to theimmunoglobulin superfamily and is expressed on T cells and pro-B cells.PD1 binds two ligands, PDL1 and PDL2. PD1, functioning as an immunecheckpoint, plays an important role in down regulating the immune systemby preventing the activation of T-cells. In certain embodiments, aligand of the composition specifically binds to PD1. In an embodiment, aligand that specifically binds to PD1 may be an anti-PD1 antibody. An“anti-PD1” includes all antibodies that specifically bind an epitopewithin PD1. The term “antibody” is described above. In anotherembodiment, a ligand that specifically binds to PD1 may be PDL1. PDL1(programmed death-ligand 1 also known as cluster of differentiation 274(CD274) or B7 homolog 1 (B7-H1), is a protein that in humans is encodedby the CD274 gene. PDL1 binds to its receptor, PD1, found on activated Tcells, B cells, and myeloid cells, to modulate activation or inhibition.The affinity between PDL1 and PD1, as defined by the dissociationconstant K_(d), is 770 nM.

In another aspect, a ligand of the composition may beGlucocorticoid-induced TNFR-related (GITR) ligand (GITRL). GITRactivation by GITRL influences the activity of effector and regulatory Tcells, thus participating in the development of immune response againsttumors and infectious agents, as well as in autoimmune and inflammatorydiseases. GITR triggering stimulates T effector activity and inhibitsTreg activity. GITR inhibition may ameliorate autoimmune/inflammatorydiseases whereas GITR activation may treat viral, bacterial andparasitic infections, as well as boost immune responses against tumors.GITRL is a type II transmembrane protein expressed at high levels onantigen presenting cells (APC) and endothelial cells.

In certain embodiments, a ligand of the invention is modified forimproved systemic half-life and reduced dosage frequency. In anembodiment, N-glycans may be added to a ligand. While the biologicalfunction is typically determined by the protein component, carbohydratecan play a role in molecular stability, solubility, in vivo activity,serum half-life, and immunogenicity. The sialic acid component ofcarbohydrate in particular, can extend the serum half-life of proteintherapeutics. Accordingly, new N-linked glycosylation consensussequences may be introduced into desirable positions in the peptidebackbone to generate proteins with increased sialic acid containingcarbohydrate, thereby increasing in vivo activity due to a longer serumhalf-life. In another embodiment, PEG may be added to a ligand. Methodsof conjugating PEG to a protein are standard in the art. For example,see Kolate et al, Journal of Controlled Release 2014; 192(28): 67-81,which is hereby incorporated by reference in its entirety. In anembodiment, a composition of the invention may comprise a ligandcomprising PEG and/or one or more N-glycans. In an embodiment, PEG isselected from the group consisting of PEG-10K, PEG-20K and PEG-40K.Still further, a ligand of the invention may be modified to remove Tcell epitopes. T cell epitopes can be the cause of an immunogenicityissue upon administration of a composition to a subject. Through theirpresentation to T cells, they activate the process of anti-drug antibodydevelopment. Preclinical screening for T cell epitopes may be performedin siico, followed by in vitro and in vivo validation. T cellepitope-mapping tools such as EpiMatrix can be highly accuratepredictors of immune response. Deliberate removal of T cell epitopes mayreduce immunogenicity. Other means of improving the safety and efficacyof a composition of the invention by reducing their immunogenicityinclude humanization and PEGylation.

(c) Linker

In an aspect, a composition of the invention further comprises a linker.The linker may be used to connect the cytokine to the ligand. It is tobe understood that linking the cytokine to the ligand will not adverselyaffect the function of the cytokine or the ligand. Suitable linkersinclude amino acid chains and alkyl chains functionalized with reactivegroups for coupling to both the cytokine and the ligand or combinationsthereof.

In an embodiment, the linker may include amino acid side chains,referred to as a peptide linker. Amino acid residue linkers are usuallyat least one residue and can be 50 or more residues, but alone do notspecifically bind to the target protein. In an embodiment, a linker maybe about 1 to about 10 amino acids. In another embodiment, a linker maybe about 10 to about 20 amino acids. In still another embodiment, alinker may be about 20 to about 30 amino acids. In still yet anotherembodiment, a linker may be about 30 to about 40 amino acids. Indifferent embodiments, a linker may be about 40 to about 50 amino acids.In other embodiments, a linker may be more than 50 amino acids. Forinstance, a linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50amino acids. In a specific embodiment, a linker is about 20 to about 30amino acids. In another specific embodiment, a linker is about 26 aminoacids.

Any amino acid residue may be used for the linker provided the linkerdoes not specifically bind to the target protein. Typical amino acidresidues used for linking are glycine, serine, alanine, leucine,tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. Forexample, a linker may be (AAS)_(n), (AAAL)_(n), (G_(n)S)_(n) or(G₂S)_(n), wherein A is alanine, S is serine, L is leucine, and G isglycine and wherein n is an integer from 1-20, or 1-10, or 3-10.Accordingly, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20. Thus, in certain embodiments, a linker includes,but is not limited to, (AAS)_(n), (AAAL)_(n), (G_(n)S)_(n) or (G₂S)_(n),wherein A is alanine, S is serine, L is leucine, and G is glycine andwherein n is an integer from 1-20, or 1-10, or 3-10. A linker maycomprise one or more epitope tags. For instance, a linker may comprise1, 2, 3, 4, 5, 6, 7 or 8 epitope tags. In a specific embodiment, alinker comprises 2 epitope tags. Non-limiting examples of epitope tagsinclude FLAG tag (DYKDDDK epitope (SEQ ID NO:9)), HA tag (YPYDVPDYAepitope (SEQ ID NO:10)), His tag (6×-His or 8×-His), Myc tag (EQKLISEEDLepitope (SEQ ID NO:11)) and V5 tag (GKPIPNPLLGLDST epitope (SEQ IDNO:12)). In an embodiment, a linker may comprise at least one tagselected from the group consisting of a FLAG tag and a His tag. In aspecific embodiment, a linker comprises a FLAG tag and a His tag. Inanother specific embodiment, a linker comprises the sequence set forthin SEQ ID NO:8 (GSSGSSDYKDDDDKHHHHHHHHGSSGSS).

In another embodiment, an alkyl chain linking group may be coupled tothe cytokine by reacting the terminal amino group or the terminalcarboxyl group with a functional group on the alkyl chain, such as acarboxyl group or an activated ester. Subsequently the ligand isattached to the alkyl chain to complete the formation of the complex byreacting a second functional group on the alkyl chain with anappropriate group on the ligand. The second functional group on thealkyl chain is selected from substituents that are reactive with afunctional group on the ligand while not being reactive with thecytokine. For example, when the ligand incorporates a functional group,such as a carboxyl group or an activated ester, the second functionalgroup of the alkyl chain linking group can be an amino group or viceversa. It will be appreciated that formation of the conjugate mayrequire protection and deprotection of the functional groups present inorder to avoid formation of undesired products. Protection anddeprotection are accomplished using protecting groups, reagents, andprotocols common in the art of organic synthesis. Particularly,protection and deprotection techniques employed in solid phase peptidesynthesis may be used. It will be appreciated that linking groups mayalternatively be coupled first to the ligand and then to the cytokine.

An alternative chemical linking group to an alkyl chain is polyethyleneglycol (PEG), which is functionalized in the same manner as the alkylchain described above. Such a linker may be referred to as aheterobifunctional PEG linker or a homobifunctional PEG linker.Non-limiting examples of heterobifunctional PEG linkers include:O-(2-Aminoethyl)-O′-[2-(biotinylamino)ethyl]octaethylene glycol;O-(2-Aminoethyl)-O′-(2-carboxyethyl)polyethylene glycol hydrochlorideM_(p) 3000; O-(2-Aminoethyl)-O′-(2-carboxyethyl)polyethylene glycol5,000 hydrochloride M_(p) 5,000; O-(2-Aminoethyl)polyethylene glycol3,000 M_(p) 3,000;O-(2-Aminoethyl)-O′-(2-(succinylamino)ethyl)polyethylene glycolhydrochloride M_(p) 10,000; O-(2-Azidoethyl)heptaethylene glycol;O-[2-(Biotinylamino)ethyl]-O′-(2-carboxyethyl)undecaethylene glycol;21-[D(+)-Biotinylamino]-4,7,10,13,16,19-hexaoxaheneicosanoic acid;O-(2-Carboxyethyl)-O′-[2-(Fmoc-amino)-ethyl]heptacosaethylene glycol;O-(2-Carboxyethyl)-O′-(2-mercaptoethyl)heptaethylene glycol;O-(3-Carboxypropyl)-O′-[2-(3-mercaptopropionylamino)ethyl]-polyethyleneglycol Mw 3000;O-(3-Carboxypropyl)-O′-[2-(3-mercaptopropionylamino)ethyl]-polyethyleneglycol Mw 5000;O-[N-(3-Maleimidopropionyl)aminoethyl]-O′-[3-(N-succinimidyloxy)-3-oxopropyl]heptacosaethyleneglycol; and O-[2-(3-Tritylthiopropionylamino)ethyl]polyethylene glycolM_(p) 3,000. Non-limiting examples of homobifunctional PEG linkersinclude: MAL-PEG-MAL (Bifunctional Maleimide PEG Maleimide);OPSS-PEG-OPSS (OPSS: orthopyridyl disulfide; PDP-PEG-PDP); HS-PEG-SH(Bifunctional Thiol PEG Thiol); SG-PEG-SG (Bifunctional PEG SuccinimidylGlutarate NHS ester); SS-PEG-SS (Bifunctional PEG Succinimidyl SuccinateNHS ester); GAS-PEG-GAS (Bifunctional PEG Succinimidyl esterNHS-PEG-NHS); SAS-PEG-SAS (Bifunctional PEG Succinimidyl esterNHS-PEG-NHS); Amine-PEG-Amine (Bifunctional PEG Amine NH2-PEG-NH2);AC-PEG-AC (Bifunctional Acrylate PEG Acrylate); ACA-PEG-ACA(Bifunctional Polymerizable PEG Acrylate Acrylamide);Epoxide-PEG-Epoxide (Bifunctional PEG Epoxide or EP); NPC-PEG-NPC(Bifunctional NPC PEG, Nitrophenyl Carbonate); Aldehyde-PEG-Aldehyde(ALD-PEG-ALD, bifunctional PEG propionaldehyde); AA-PEG-AA(Acid-PEG-Acid, AA-acetic acid or carboxyl methyl); GA-PEG-GA(Acid-PEG-Acid, GA: Glutaric acid); SA-PEG-SA (Bifunctional PEGcarboxylic acid-Succinic Acid); GAA-PEG-GAA (Bifunctional PEG carboxylicacid, Glutaramide Acid); SAA-PEG-SAA (Bifunctional PEG carboxylic acid,Succinamide Acid); Azide-PEG-Azide (Bifunctional PEG azide, N3-PEG-N3);Alkyne-PEG-Alkyne (Bifunctional alkyne or acetylene PEG);Biotin-PEG-Biotin (Bifunctional biotin PEG linker); Silane-PEG-Silane(Bifunctional silane PEG); Hydrazide-PEG-Hydrazide (Bifunctional PEGHydrazide); Tosylate-PEG-Tosylate (Bifunctional PEG Tosyl); andChloride-PEG-Chloride (Bifunctional PEG Halide).

In certain embodiments, a linker of the invention may be modified forimproved systemic half-life and reduced dosage frequency. In anembodiment, N-glycans are added to a linker. While the biologicalfunction is typically determined by the protein component, carbohydratescan play a role in molecular stability, solubility, in vivo activity,serum half-life, and immunogenicity. The sialic acid component ofcarbohydrate in particular, can extend the serum half-life of proteintherapeutics. Accordingly, new N-linked glycosylation consensussequences may be introduced into desirable positions in the peptidebackbone to generate proteins with increased sialic acid containingcarbohydrate, thereby increasing in vivo activity due to a longer serumhalf-life. In another embodiment, PEG is added to a linker. Methods ofconjugating PEG to a protein are standard in the art. For example, seeKolate et al, Journal of Controlled Release 2014; 192(28): 67-81, whichis hereby incorporated by reference in its entirety. In an embodiment, acomposition of the invention comprises a ligand comprising PEG and/orone or more N-glycans. In an embodiment, PEG is selected from the groupconsisting of PEG-10K, PEG-20K and PEG-40K.

Another aspect of the invention involves cross-linking the peptides ofthe invention to improve their pharmacokinetic, immunogenic, diagnostic,and/or therapeutic attributes. Cross-linking involves joining twomolecules by a covalent bond through a chemical reaction at suitablesite(s) (e.g., primary amines, sulfhydryls) on the cytokine and ligandof the invention. In an embodiment, the cytokine and ligand may becross-linked together. The cross-linking agents may form a cleavable ornon-cleavable linker between the cytokine and the ligand. Cross-linkingagents that form non-cleavable linkers between the cytokine and theligand may comprise a maleimido- or haloacetyl-based moiety. Accordingto the present invention, such non-cleavable linkers are said to bederived from maleimido- or haloacetyl-based moiety. Cross-linking agentscomprising a maleimido-based moiety include N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC), K-maleimidoundecanoicacid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidylester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-(α-maleimidoacetoxy)-succinimide ester [AMAS],succinimidyl-6-(3-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl4-(α-maleimidophenyl)-butyrate (SMPB), andN-(p-maleimidophenyl)isocyanate (PMPI). These cross-linking agents formnon-cleavable linkers derived from maleimido-based moieties.Cross-linking agents comprising a haloacetyl-based moiety includeN-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyliodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl3-(bromoacetamido)propionate (SBAP). These cross-linking agents formnon-cleavable linkers derived from haloacetyl-based moieties.Cross-linking agents that form non-cleavable linkers between thecytokine and the ligand may comprise N-succinimidyl3-(2-pyridyldithio)propionate,4-succinimidyl-oxycarbonyl-α-methyl-alpha-(2-pyridyldithio)-toluene(SMPT), N-succinimidyl-3-(2-pyridyldithio)-butyrate (SDPB),2-iminothiolane, or acetylsuccinic anhydride.

(d) Chimeric Peptide

In another aspect, the invention encompasses a chimeric peptidecomprising a cytokine peptide and a ligand peptide, wherein the cytokinepeptide is not a binding partner of the ligand peptide. In still anotheraspect, the invention encompasses a chimeric peptide comprising acytokine peptide and a ligand peptide, wherein the ligand peptide bindsto an NKG2D receptor. It should be understood that “ligand peptideis”used interchangeably with “liganda” and “cytokine peptide” is usedinterchangeably with “cytokine” for purposes of descriptions herein ofvarious cytokines and ligands that are suitable for use in the presentcompositions and methods.

In certain embodiments, the cytokine peptide is in the IL2 subfamily.More specifically, the cytokine peptide is selected from the groupconsisting of IL2, IL7, IL15 and IL21. In a specific embodiment, thecytokine peptide is IL15 or a mutant thereof. In another specificembodiment, the cytokine peptide is IL2 or a mutant thereof. In anotherembodiment, the cytokine peptide is mutant IL2 comprising at least onemutation selected from the group consisting of R38A, F42K and C125S. Ina specific embodiment, the cytokine peptide comprises the amino acidsequence set forth in SEQ ID NO:5 or SEQ ID NO:6. In other embodiments,the cytokine peptide is in the IL1 family. More specifically, thecytokine peptide is selected from the group consisting of IL1α, IL1β,IL1Ra, IL18, IL36Ra, IL36α, IL37, IL36β, IL36γ, IL38, and IL33. In aspecific embodiment, the cytokine peptide is IL18 or a mutant thereof.

In certain embodiments, the ligand peptide is a MHC class-I-relatedglycoprotein. In another embodiment, the ligand peptide is OMCP, aportion thereof, or a mutant thereof. In an embodiment, the ligandpeptide binds to a receptor expressed on NK cells and CD8+ CTLs. In aspecific embodiment, the ligand peptide binds to an NKG2D receptor. Incertain embodiments, the ligand peptide comprises the amino acidsequence set forth in SEQ ID NO:7 or a portion thereof that is capableof binding to the NKG2D receptor.

In other embodiments, a chimeric peptide further comprises a linkerpeptide. In certain embodiments, a linker peptide comprises the aminoacid sequence selected from the group consisting of (AAS)_(n),(AAAL)_(n), (G_(n)S)_(n) or (G₂S)_(n), wherein A is alanine, S isserine, L is leucine, and G is glycine and wherein n is an integer from1-20, or 1-10, or 3-10. In a different embodiment, a linker peptidecomprises at least one tag selected from the group consisting of a FLAGtag and a His tag. In an embodiment, a linker peptide is about 20 toabout 30 amino acids. In a specific embodiment, a linker peptidecomprises the amino acid sequence set forth in SEQ ID NO:8.

The invention also encompasses a nucleic acid molecule encoding achimeric peptide as described herein. Additionally, the inventionencompasses a pharmaceutical composition comprising a chimeric peptideas described herein. Pharmaceutical compositions are described in moredetail in Section I(f).

(e) Preferred Embodiments

By way of non-limiting example, several preferred compositions of theinvention are depicted in FIG. 23. 1. depicts a composition comprisingα2 domain (H2) of OMCP linked to a cytokine. 2. depicts a compositioncomprising OMCP linked to a cytokine, wherein the composition ispegylated. 3. depicts a composition comprising OMCP linked to acytokine, wherein the composition comprises N-glycan. 4. depicts acomposition comprising, OMCP linked to a cytokine, wherein the linkercomprises various sequences and various lengths. 5. depicts acomposition comprising a Fab specific antibody for NKG2D linked to acytokine. 6. depicts a composition comprising various NKG2D ligandslinked to a cytokine. 7. depicts a composition comprising a mutatedversion of OMCP linked to a cytokine, wherein the OMCP may be mutated tohave improved binding affinity or weaker binding affinity. 8. depicts acomposition comprising a mutated version of OMCP linked to a cytokine,wherein the OMCP may be mutated to have binding affinity for other NKG2receptors. 9. depicts a composition comprising a viral protein liked toa cytokine. For example, OMCP binds to NKG2D. Additionally, CPXV203binds to MHCI. 10. depicts a composition comprising OMCP linked to amutated cytokine. It is understood that the OMCP sequence could be fromvarious sources such as cowpox or monkeypox. Also, Fc-chimeras of OMCPand IL2, and variants thereof may be used.

In a preferred embodiment, the composition comprises IL2, IL15 or IL18linked to OMCP. In another preferred embodiment, the compositioncomprises IL2, IL15 or IL18 linked to OMCP via a peptide linker. Instill another preferred embodiment, the composition comprises IL2, IL15or IL18 linked to OMCP via a peptide linker comprising about 20 to about30 amino acids. In still yet another preferred embodiment, thecomposition comprises IL2, IL15 or IL18 linked to OMCP via a peptidelinker comprising a FLAG tag and a His tag. In each of the foregoingembodiments, the IL2 may be a mutated version of IL2 comprising themutations R38A and F42K.

In a different preferred embodiment, the composition comprises IL2linked to OMCP. In another preferred embodiment, the compositioncomprises IL2 linked to OMCP via a peptide linker. In still anotherpreferred embodiment, the composition comprises IL2 linked to OMCP via apeptide linker comprising about 20 to about 30 amino acids. In still yetanother preferred embodiment, the composition comprises IL2 linked toOMCP via a peptide linker comprising a FLAG tag and a His tag. In eachof the foregoing embodiments, the IL2 may be a mutated version of IL2comprising the mutations R38A and F42K.

In an exemplary embodiment, the compositioncomprises the DNA sequence set forth in SEQ ID NO: 1(CACAAACTCGCATTCAACTTCAATCTAGAAATAAATGGCAGTGATACACATTCTACAGTAGATGTATATCTTGATGATTCTCAAATTATAACGTTTGATGGAAAAGATATCCGTCCAACCATCCCGTTCATGATAGGTGATGAAATTTTCTTACCGTTTTATAAAAATGTGTTTAGTGAGTTTTTCTCTCTGTTTAGAAGAGTTCCTACAAGTACTCCATATGAAGACTTGACATATTTTTATGAATGCGACTATACAGACAATAAATCTACATTTGATCAGTTTTATCTTTATAATGGCGAAGAATATACTGTCAAAACACAGGAGGCCACTAATAAAAATATGTGGCTAACTACTTCCGAGTTTAGACTAAAAAAATGGTTCGATGGCGAAGATTGTATAATGCATCTTAGATCGTTAGTTAGAAAAATGGAGGACAGTAAACGAAACACTGGTGGTACCGGAAGTAGCGGTAGTAGTGATTACAAGGACGATGACGACAAGCACCACCATCATCATCATCACCACGGTAGCAGCGGCAGCAGTGCCCCCACCTCTAGCAGCACAAAGAAGACCCAGCTGCAACTGGAACACCTCCTGCTGGACCTGCAGATGATCCTGAACGGCATCAACAACTACAAGAACCCCAAGCTGACCGCCATGCTGACCAAAAAGTTTTACATGCCCAAGAAGGCCACCGAGCTTAAACACCTGCAATGCCTTGAGGAGGAGCTGAAGCCCTGGAGGAGGTACTGAACCTGGCCCAGAGCAAGAACTTTCATCTGAGGCCCAGGGACCTGATTAGCAACATCAACGTGATCGTGTTGGAGTTGAAGGGCAGCGAGACCACGTTCATGTGCGAGTACGCCGACGAGACGGCCACCATAGTGGAGTTTCTTAACAGGTGGATCACCTTCTCACAGTCTATCATCAGCACCCTGACC).In another exemplary embodiment, the compositioncomprises the amino acid sequence set forth in SEQ ID NO: 2(HKLAFNFNLEINGSDTHSTVDVYLDDSQIITFDGKDIRPTIPFMIGDEIFLPFYKNVFSEFFSLFRRVPTSTPYEDLTYFYECDYTDNKSTFDQFYLYNGEEYTVKTQEATNKNMWLTTSEFRLKKWFDGEDCIMHLRSLVRKMEDSKRNTGGTGSSGSSDYKDDDDKHHHHHHHHGSSGSSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF LNRWITFSQSIISTLT).

(f) Pharmaceutical Compositions

The present disclosure also provides pharmaceutical compositions. Thepharmaceutical composition comprises a composition of the inventionwhich is detailed above, as an active ingredient and at least onepharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient may be a diluent, a binder, afiller, a buffering agent, a pH modifying agent, a disintegrant, adispersant, a preservative, a lubricant, taste-masking agent, aflavoring agent, or a coloring agent. The amount and types of excipientsutilized to form pharmaceutical compositions may be selected accordingto known principles of pharmaceutical science.

In one embodiment, the excipient may be a diluent. The diluent may becompressible (i.e., plastically deformable) or abrasively brittle.Non-limiting examples of suitable compressible diluents includemicrocrystalline cellulose (MCC), cellulose derivatives, cellulosepowder, cellulose esters (i.e., acetate and butyrate mixed esters),ethyl cellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, sodium carboxymethylcellulose, cornstarch, phosphated corn starch, pregelatinized corn starch, rice starch,potato starch, tapioca starch, starch-lactose, starch-calcium carbonate,sodium starch glycolate, glucose, fructose, lactose, lactosemonohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol,xylitol, maltodextrin, and trehalose. Non-limiting examples of suitableabrasively brittle diluents include dibasic calcium phosphate (anhydrousor dihydrate), calcium phosphate tribasic, calcium carbonate, andmagnesium carbonate.

In another embodiment, the excipient may be a binder. Suitable bindersinclude, but are not limited to, starches, pregelatinized starches,gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylamides,polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohol,polyethylene glycol, polyols, saccharides, oligosaccharides,polypeptides, oligopeptides, and combinations thereof.

In another embodiment, the excipient may be a filler. Suitable fillersinclude, but are not limited to, carbohydrates, inorganic compounds, andpolyvinylpyrrolidone. By way of non-limiting example, the filler may becalcium sulfate, both di- and tri-basic, starch, calcium carbonate,magnesium carbonate, microcrystalline cellulose, dibasic calciumphosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc,modified starches, lactose, sucrose, mannitol, or sorbitol.

In still another embodiment, the excipient may be a buffering agent.Representative examples of suitable buffering agents include, but arenot limited to, phosphates, carbonates, citrates, tris buffers, andbuffered saline salts (e.g., Tris buffered saline or phosphate bufferedsaline).

In various embodiments, the excipient may be a pH modifier. By way ofnon-limiting example, the pH modifying agent may be sodium carbonate,sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

In a further embodiment, the excipient may be a disintegrant. Thedisintegrant may be non-effervescent or effervescent. Suitable examplesof non-effervescent disintegrants include, but are not limited to,starches such as corn starch, potato starch, pregelatinized and modifiedstarches thereof, sweeteners, clays, such as bentonite,micro-crystalline cellulose, alginates, sodium starch glycolate, gumssuch as agar, guar, locust bean, karaya, pecitin, and tragacanth.Non-limiting examples of suitable effervescent disintegrants includesodium bicarbonate in combination with citric acid and sodiumbicarbonate in combination with tartaric acid.

In yet another embodiment, the excipient may be a dispersant ordispersing enhancing agent. Suitable dispersants may include, but arenot limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum,kaolin, bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose.

In another alternate embodiment, the excipient may be a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate,citric acid, sodium citrate; chelators such as EDTA or EGTA; andantimicrobials, such as parabens, chlorobutanol, or phenol.

In a further embodiment, the excipient may be a lubricant. Non-limitingexamples of suitable lubricants include minerals such as talc or silica;and fats such as vegetable stearin, magnesium stearate or stearic acid.

In yet another embodiment, the excipient may be a taste-masking agent.Taste-masking materials include cellulose ethers; polyethylene glycols;polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers;monoglycerides or triglycerides; acrylic polymers; mixtures of acrylicpolymers with cellulose ethers; cellulose acetate phthalate; andcombinations thereof.

In an alternate embodiment, the excipient may be a flavoring agent.Flavoring agents may be chosen from synthetic flavor oils and flavoringaromatics and/or natural oils, extracts from plants, leaves, flowers,fruits, and combinations thereof.

In still a further embodiment, the excipient may be a coloring agent.Suitable color additives include, but are not limited to, food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drugand cosmetic colors (Ext. D&C).

The weight fraction of the excipient or combination of excipients in thecomposition may be about 99% or less, about 97% or less, about 95% orless, about 90% or less, about 85% or less, about 80% or less, about 75%or less, about 70% or less, about 65% or less, about 60% or less, about55% or less, about 50% or less, about 45% or less, about 40% or less,about 35% or less, about 30% or less, about 25% or less, about 20% orless, about 15% or less, about 10% or less, about 5% or less, about 2%,or about 1% or less of the total weight of the composition.

The composition can be formulated into various dosage forms andadministered by a number of different means that will deliver atherapeutically effective amount of the active ingredient. Suchcompositions can be administered orally, parenterally, or topically indosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants, and vehicles asdesired. Topical administration may also involve the use of transdermaladministration such as transdermal patches or iontophoresis devices. Theterm parenteral as used herein includes subcutaneous, intravenous,intramuscular, or intrasternal injection, or infusion techniques.Formulation of drugs is discussed in, for example, Gennaro, A. R.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.(18^(th) ed, 1995), and Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980).

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, powders, pellets, and granules. In such solid dosageforms, the active ingredient is ordinarily combined with one or morepharmaceutically acceptable excipients, examples of which are detailedabove. Oral preparations may also be administered as aqueoussuspensions, elixirs, or syrups. For these, the active ingredient may becombined with various sweetening or flavoring agents, coloring agents,and, if so desired, emulsifying and/or suspending agents, as well asdiluents such as water, ethanol, glycerin, and combinations thereof.

For parenteral administration (including subcutaneous, intradermal,intravenous, intramuscular, and intraperitoneal), the preparation may bean aqueous or an oil-based solution. Aqueous solutions may include asterile diluent such as water, saline solution, a pharmaceuticallyacceptable polyol such as glycerol, propylene glycol, or other syntheticsolvents; an antibacterial and/or antifungal agent such as benzylalcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and thelike; an antioxidant such as ascorbic acid or sodium bisulfite; achelating agent such as etheylenediaminetetraacetic acid; a buffer suchas acetate, citrate, or phosphate; and/or an agent for the adjustment oftonicity such as sodium chloride, dextrose, or a polyalcohol such asmannitol or sorbitol. The pH of the aqueous solution may be adjustedwith acids or bases such as hydrochloric acid or sodium hydroxide.Oil-based solutions or suspensions may further comprise sesame, peanut,olive oil, or mineral oil.

For topical (e.g., transdermal or transmucosal) administration,penetrants appropriate to the barrier to be permeated are generallyincluded in the preparation. Transmucosal administration may beaccomplished through the use of nasal sprays, aerosol sprays, tablets,or suppositories, and transdermal administration may be via ointments,salves, gels, patches, or creams as generally known in the art.

In certain embodiments, a composition comprising a compound of theinvention is encapsulated in a suitable vehicle to either aid in thedelivery of the compound to target cells, to increase the stability ofthe composition, or to minimize potential toxicity of the composition.As will be appreciated by a skilled artisan, a variety of vehicles aresuitable for delivering a composition of the present invention.Non-limiting examples of suitable structured fluid delivery systems mayinclude nanoparticles, liposomes, microemulsions, micelles, dendrimersand other phospholipid-containing systems. Methods of incorporatingcompositions into delivery vehicles are known in the art.

In one alternative embodiment, a liposome delivery vehicle may beutilized. Liposomes, depending upon the embodiment, are suitable fordelivery of the compound of the invention in view of their structuraland chemical properties. Generally speaking, liposomes are sphericalvesicles with a phospholipid bilayer membrane. The lipid bilayer of aliposome may fuse with other bilayers (e.g., the cell membrane), thusdelivering the contents of the liposome to cells. In this manner, thecompound of the invention may be selectively delivered to a cell byencapsulation in a liposome that fuses with the targeted cell'smembrane.

Liposomes may be comprised of a variety of different types ofphospholipids having varying hydrocarbon chain lengths. Phospholipidsgenerally comprise two fatty acids linked through glycerol phosphate toone of a variety of polar groups. Suitable phospholipids includephosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol(PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG),phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fattyacid chains comprising the phospholipids may range from about 6 to about26 carbon atoms in length, and the lipid chains may be saturated orunsaturated. Suitable fatty acid chains include (common name presentedin parentheses) n-dodecanoate (laurate), n-tretradecanoate (myristate),n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate(arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate),cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate),cis,cis-9,12-octadecandienoate (linoleate), all cis-9, 12,15-octadecatrienoate (linolenate), and allcis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acidchains of a phospholipid may be identical or different. Acceptablephospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS,distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl,oleoyl PS, palmitoyl, linolenyl PS, and the like.

The phospholipids may come from any natural source, and, as such, maycomprise a mixture of phospholipids. For example, egg yolk is rich inPC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brainor spinal cord is enriched in PS. Phospholipids may come from syntheticsources too. Mixtures of phospholipids having a varied ratio ofindividual phospholipids may be used. Mixtures of differentphospholipids may result in liposome compositions having advantageousactivity or stability of activity properties. The above mentionedphospholipids may be mixed, in optimal ratios with cationic lipids, suchas N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride,1,1′-dioctadecyl-3,3,3′, 3′-tetramethylindocarbocyanine perchloarate,3,3′-deheptyloxacarbocyanine iodide, 1,1′-dedodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate, 1,1′-dioleyl-3,3,3′,3′-tetramethylindo carbocyanine methanesulfonate,N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or1,1,-dilinoleyl-3,3,3′, 3′-tetramethylindocarbocyanine perchloarate.

Liposomes may optionally comprise sphingolipids, in which spingosine isthe structural counterpart of glycerol and one of the one fatty acids ofa phosphoglyceride, or cholesterol, a major component of animal cellmembranes. Liposomes may optionally, contain pegylated lipids, which arelipids covalently linked to polymers of polyethylene glycol (PEG). PEGsmay range in size from about 500 to about 10,000 daltons.

Liposomes may further comprise a suitable solvent. The solvent may be anorganic solvent or an inorganic solvent. Suitable solvents include, butare not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone,N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide,tetrahydrofuran, or combinations thereof.

Liposomes carrying the compound of the invention (i.e., having at leastone methionine compound) may be prepared by any known method ofpreparing liposomes for drug delivery, such as, for example, detailed inU.S. Pat. Nos. 4,241,046, 4,394,448, 4,529,561, 4,755,388, 4,828,837,4,925,661, 4,954,345, 4,957,735, 5,043,164, 5,064,655, 5,077,211 and5,264,618, the disclosures of which are hereby incorporated by referencein their entirety. For example, liposomes may be prepared by sonicatinglipids in an aqueous solution, solvent injection, lipid hydration,reverse evaporation, or freeze drying by repeated freezing and thawing.In a preferred embodiment the liposomes are formed by sonication. Theliposomes may be multilamellar, which have many layers like an onion, orunilamellar. The liposomes may be large or small. Continued high-shearsonication tends to form smaller unilamellar lipsomes.

As would be apparent to one of ordinary skill, all of the parametersthat govern liposome formation may be varied. These parameters include,but are not limited to, temperature, pH, concentration of methioninecompound, concentration and composition of lipid, concentration ofmultivalent cations, rate of mixing, presence of and concentration ofsolvent.

In another embodiment, a composition of the invention may be deliveredto a cell as a microemulsion. Microemulsions are generally clear,thermodynamically stable solutions comprising an aqueous solution, asurfactant, and “oil.” The “oil” in this case, is the supercriticalfluid phase. The surfactant rests at the oil-water interface. Any of avariety of surfactants are suitable for use in microemulsionformulations including those described herein or otherwise known in theart. The aqueous microdomains suitable for use in the inventiongenerally will have characteristic structural dimensions from about 5 nmto about 100 nm. Aggregates of this size are poor scatterers of visiblelight and hence, these solutions are optically clear. As will beappreciated by a skilled artisan, microemulsions can and will have amultitude of different microscopic structures including sphere, rod, ordisc shaped aggregates. In one embodiment, the structure may bemicelles, which are the simplest microemulsion structures that aregenerally spherical or cylindrical objects. Micelles are like drops ofoil in water, and reverse micelles are like drops of water in oil. In analternative embodiment, the microemulsion structure is the lamellae. Itcomprises consecutive layers of water and oil separated by layers ofsurfactant. The “oil” of microemulsions optimally comprisesphospholipids. Any of the phospholipids detailed above for liposomes aresuitable for embodiments directed to microemulsions. The composition ofthe invention may be encapsulated in a microemulsion by any methodgenerally known in the art.

In yet another embodiment, a composition of the invention may bedelivered in a dendritic macromolecule, or a dendrimer. Generallyspeaking, a dendrimer is a branched tree-like molecule, in which eachbranch is an interlinked chain of molecules that divides into two newbranches (molecules) after a certain length. This branching continuesuntil the branches (molecules) become so densely packed that the canopyforms a globe. Generally, the properties of dendrimers are determined bythe functional groups at their surface. For example, hydrophilic endgroups, such as carboxyl groups, would typically make a water-solubledendrimer. Alternatively, phospholipids may be incorporated in thesurface of a dendrimer to facilitate absorption across the skin. Any ofthe phospholipids detailed for use in liposome embodiments are suitablefor use in dendrimer embodiments. Any method generally known in the artmay be utilized to make dendrimers and to encapsulate compositions ofthe invention therein. For example, dendrimers may be produced by aniterative sequence of reaction steps, in which each additional iterationleads to a higher order dendrimer.

Consequently, they have a regular, highly branched 3D structure, withnearly uniform size and shape. Furthermore, the final size of adendrimer is typically controlled by the number of iterative steps usedduring synthesis. A variety of dendrimer sizes are suitable for use inthe invention. Generally, the size of dendrimers may range from about 1nm to about 100 nm

II. Methods

In an aspect, the invention encompasses a method to deliver a cytokineto a target cell. The method comprises contacting a target cell with acomposition comprising a cytokine linked to a ligand, wherein the ligandspecifically binds to a receptor on the target cell. Additionally, themethod comprises contacting a target cell with a composition comprisinga chimeric peptide as described in Section I. A target cell may be anycell comprising a target receptor for which the ligand specificallybinds to. The ligand and specific binding are described in Section I. Incertain embodiments, a target cell may be an immune cell. Non-limitingexample of immune cells include macrophages, B lymphocytes, Tlymphocytes, mast cells, monocytes, dendritic cells, eosinophils,natural killer cells, basophils, neutrophils. In certain embodiments, animmune cell is selected from the group consisting of a macrophage, Blymphocyte, T lymphocyte, mast cell, monocyte, dendritic cell,eosinophil, natural killer cell, basophil, and neutrophil. In a specificembodiment, a target cell is a natural killer (NK) cell and/or a CD8+ Tcell. In other embodiments, a target cell is a NKG2D-expressing cell.Non-limiting examples of NKG2D-expressing cell include natural killer(NK) cells and CD8+ T cells (both αβ and γδ).

In another aspect, the invention encompasses a method to activate immunecells. The method comprises contacting an immune cell with a compositioncomprising a cytokine linked to a ligand, wherein the ligandspecifically binds to a receptor on the immune cell thereby activatingthe cell. Additionally, the method comprises contacting an immune cellwith a composition comprising a chimeric peptide as described in SectionI. Non-limiting example of immune cells include macrophages, Blymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells,eosinophils, natural killer cells, basophils, neutrophils. In certainembodiments, an immune cell is selected from the group consisting of amacrophage, B lymphocyte, T lymphocyte, mast cell, monocyte, dendriticcell, eosinophil, natural killer cell, basophil, and neutrophil. In aspecific embodiment, an immune cell is a natural killer (NK) cell and/ora CD8+ T cell. To facilitate activation of immune cells, a cytokine maybe a proinflammatory cytokine. The term “proinflammatory cytokine” is acytokine which promotes systemic inflammation. A skilled artisan wouldbe able to determine those cytokines that are proinflammatory. Incertain embodiments, a proinflammatory cytokine is IL1α, IL1β, IL2, IL3,IL6, IL7, IL9, IL12, IL15, IL17, IL18, IL21, IFNα, IFNγ, TNFα, MIF,G-CSF, GM-CSF or mutants thereof. In an embodiment, a proinflammatorycytokine is an IL1 family cytokine. In certain embodiments, an IL1family cytokine is selected from the group consisting of IL1α, IL1β,IL1Ra, IL18, IL36Ra, IL36α, IL37, IL36β, IL36γ, IL38, IL33 and mutantsthereof. In a specific embodiment, a proinflammatory cytokine isselected from the group consisting of IL2, IL7, IL15, IL18, IL21 andmutants thereof. In another specific embodiment, a proinflammatorycytokine is selected from the group consisting of IL2, IL15, IL18, andmutants thereof. In an exemplary embodiment, a proinflammatory cytokineis IL2 or a mutant thereof. Activation of the immune cells may result inlysis of tumor cells. Accordingly, activation of immune cells may bemeasured by determining the amount of tumor cell lysis. In anembodiment, activation of the immune cells may result in about 10% toabout 100% lysis of tumor cells. In another embodiment, activation ofthe immune cells may result in about 20% to about 80% lysis of tumorcells. In still another embodiment, activation of the immune cells mayresult in greater than 40% lysis of tumor cells. For example, activationof the immune cells may result in greater than 40%, greater than 45%,greater than 50%, greater than 55%, greater than 60%, greater than 65%,greater than 70%, greater than 75%, greater than 80%, greater than 85%,greater than 90%, greater than 95%, or greater than 99% lysis of tumorcells. The lysis of tumor cells may be measured using any standard assay(e.g., caspase assays, TUNEL and DNA fragmentation assays, cellpermeability assays, and Annexin V assays).

In still another aspect, the invention encompasses a method to treat atumor. The method comprises identifying a subject with a tumor andadministering to the subject a composition comprising a cytokine linkedto a ligand, wherein the ligand specifically binds to a receptor on atarget cell. Additionally, the method comprises administering to thesubject a composition comprising a chimeric peptide as described inSection I. Specifically, the inventors have shown that delivering acytokine to a target cell activates the cells bound by the composition,wherein the activated cells specifically lyse tumor cells therebyreducing the amount of cancer cells. In a specific embodiment, acytokine is a proinflammatory cytokine as described in the precedingparagraph. Accordingly, a composition of the present invention, may beused in treating, stabilizing and preventing cancer and associateddiseases in a subject. By “treating, stabilizing, or preventing cancer”is meant causing a reduction in the size of a tumor or in the number ofcancer cells, slowing or preventing an increase in the size of a tumoror cancer cell proliferation, increasing the disease-free survival timebetween the disappearance of a tumor or other cancer and itsreappearance, preventing an initial or subsequent occurrence of a tumoror other cancer, or reducing an adverse symptom associated with a tumoror other cancer. The inventors have shown that a composition of theinvention activates natural killer (NK) cells bound by the composition,wherein the activated NK cells specifically lyse tumor cells therebyreducing the amount of tumor cells. For example, as cancerous cells are“stressed”, NKG2D ligands become upregulated, rendering the cellsusceptible to NK cell-mediated lysis. In a desired embodiment, thepercent of tumor or cancerous cells surviving the treatment is at least20, 30, 40, 50, 60, 70, 80, 90 or 100% lower than the initial number oftumor or cancerous cells, as measured using any standard assay (e.g.,caspase assays, TUNEL and DNA fragmentation assays, cell permeabilityassays, and Annexin V assays). Desirably, the decrease in the number oftumor or cancerous cells induced by administration of a composition ofthe invention is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45 or50-fold greater than the decrease in the number of non-tumor ornon-cancerous cells. Desirably, the methods of the present inventionresult in a decrease of 20, 30, 40, 50, 60, 50, 80, 90 or 100% in thesize of a tumor or in the number of cancerous cells, as determined usingstandard methods. Desirably, at least 20, 30, 40, 50, 60, 70, 80, 90, or95% of the treated subjects have a complete remission in which allevidence of the tumor or cancer disappears. Desirably, the tumor orcancer does not reappear or reappears after at least 1, 2, 3, 4, 5, 10,15, or 20 years.

In another aspect, the invention encompasses a method to suppress immunecells. The method comprises contacting an immune cell with a compositioncomprising a cytokine linked to a ligand, wherein the ligandspecifically binds to a receptor on the immune cell thereby suppressingthe cell. Additionally, the method comprises contacting an immune cellswith a composition comprising a chimeric peptide as described in SectionI. Non-limiting example of immune cells include macrophages, Blymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells,eosinophils, natural killer cells, basophils, neutrophils. In certainembodiments, an immune cell is selected from the group consisting of amacrophage, B lymphocyte, T lymphocyte, mast cell, monocyte, dendriticcell, eosinophil, natural killer cell, basophil, and neutrophil. In aspecific embodiment, an immune cell is a natural killer (NK) cell and/ora CD8+ T cell. To facilitate suppression of immune cells, a cytokine maybe an anti-inflammatory cytokine. The term “anti-inflammatory cytokine”is a cytokine that counteracts various aspects of inflammation, forexample cell activation or the production of proinflammatory cytokines,and thus contributes to the control of the magnitude of the inflammatoryresponse. A skilled artisan would be able to determine those cytokinesthat are anti-inflammatory. In certain embodiments, an anti-inflammatorycytokine is IL4, IL5, IL10, IL11, IL13, IL16, IL35, IFNα, TGFβ, G-CSF ora mutant thereof. In a specific embodiment, an anti-inflammatorycytokine is IL10 or a mutant thereof. In another embodiment, theinvention encompasses a method to kill immune cells. The methodcomprises contacting an immune cell with a composition comprising atoxin linked to a ligand, wherein the ligand specifically binds to areceptor on the immune cell thereby killing the cell. Suppression orkilling of the immune cells may result in treatment, stabilization andprevention of autoimmune diseases caused by overactive immune cells.NKG2D-expressing cells and/or aberrant expression of host NKG2DLs havebeen implicated in diabetes, celiac disease and rheumatoid arthritis.For example, NK cells can recognize pancreatic beta cells and destroythem. The destruction of pancreatic beta cells may lead to type 1diabetes. By way of another example, overactive immune cells areinvolved in transplant/graft rejection. Accordingly, a composition ofthe present invention, may be used in treating, stabilizing andpreventing an automimmune disease in a subject. In a specificembodiment, the autoimmune disease is type 1 diabetes. In anotherspecific embodiment, the autoimmune disease is transplant or graftrejection. In still another specific embodiment, the autoimmune diseaseis rheumatoid arthritis.

In still yet another aspect, the invention encompasses a method to treatan infection comprising administering a composition comprising acytokine linked to a ligand. For example, a composition comprising acytokine linked to a ligand may specifically bind an immune cell that isthen activated to target and lyse the infected host cell. Additionally,the method comprises administering to the subject a compositioncomprising a chimeric peptide as described in Section I. The term“infection” as used herein includes the presence of pathogens in or on asubject, which, if its growth were inhibited, would result in a benefitto the subject. As such, the term “infection” in addition to referringto the presence of pathogens also refers to normal flora which are notdesirable. The term “pathogen” as used herein refers to an infectiousagent that can produce disease. Non-limiting examples of an infectiousagent include virus, bacterium, prion, fungus, viroid, or parasite thatcause disease in a subject. In a specific embodiment, an infection iscaused by pathogens such as bacteria or viruses. In certain embodiments,the infection is an intracellular infection. In an embodiment, theinfection is a viral infection. In another embodiment, the viralinfection is caused by a flavivirus. Flavivirus is a genus of viruses inthe family Flaviviridae. Non-limiting examples of flaviviruses includeGadget's Gully virus, Kadam virus, Kyasanur Forrest disease virus,Langat virus, Omsk hemorrhagic fever virus, Tick-borne encephalitisvirus, Louping ill virus, Aroa virus, Dengue viruses 1-4, Kedougouvirus, Cacipacore virus, Koutango virus, Murray Valley encephalitisvirus, St. Louis encephalitis virus, Usutu virus, West Nile virus,Yaounde virus, Kokobera virus group, Kokobera virus, Bagaza virus,Ilheus virus, Israel turkey meningoencephalomyelitis virus, Ntaya virus,Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge Hill virus,Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Wesselsbronvirus, Yellow fever virus, Entebbe bat virus, Yokose virus, Apoi virus,Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, SanPerlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat virus,Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravovirus, hepatitis C virus, e.g., hepatitis C virus genotypes 1-6, and GBvirus A and B. In a certain embodiment, the flavivirus may be selectedfrom the group consisting of West Nile virus, dengue virus, Japaneseencephalitis virus, and yellow fever virus. In a specific embodiment,the viral infection is caused by West Nile virus. In certainembodiments, a pathogen, more specifically a virus, can induce theexpression of proteins for which NKG2D binds. Accordingly, a compositioncomprising a cytokine linked to a ligand may specifically bind a NK cellthat is then activated to target and lyse the infected host cellexpressing NKG2D. In another embodiment, a composition comprising acytokine linked to a ligand may activate cytotoxic T lymphocytes thatrecognize infected cells via other mechanisms for targeted killing.

In a different aspect, the invention encompasses a method to alleviateimmunosuppression related to radiation exposure or lympotoxic substancescomprising administering a composition comprising a cytokine linked to aligand. Additionally, the method comprises administering a compositioncomprising a chimeric peptide as described in Section I. Additionally, acomposition of the invention may be used to raise CD4 counts in HIVpositive subjects. For example, a composition of the invention may beused to activate immune cells which can help restore the immune systemof the subject.

In an alternative aspect, the invention encompasses a method of use asan adjuvant in a vaccine composition. For example, a composition of theinvention may be used to expand CD8+ memory cells.

(a) Administration

In certain aspects, a pharmacologically effective amount of acomposition of the invention may be administered to a subject.Administration is performed using standard effective techniques,including peripherally (i.e. not by administration into the centralnervous system) or locally to the central nervous system. Peripheraladministration includes but is not limited to intravenous,intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular,intranasal, buccal, sublingual, or suppository administration. Localadministration, including directly into the central nervous system (CNS)includes but is not limited to via a lumbar, intraventricular orintraparenchymal catheter or using a surgically implanted controlledrelease formulation. Pheresis may be used to deliver a composition ofthe invention. In certain embodiments, a composition of the inventionmay be administered via an infusion (continuous or bolus).

Pharmaceutical compositions for effective administration aredeliberately designed to be appropriate for the selected mode ofadministration, and pharmaceutically acceptable excipients such ascompatible dispersing agents, buffers, surfactants, preservatives,solubilizing agents, isotonicity agents, stabilizing agents and the likeare used as appropriate. Remington's Pharmaceutical Sciences, MackPublishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition,incorporated herein by reference in its entirety, provides a compendiumof formulation techniques as are generally known to practitioners.

Effective peripheral systemic delivery by intravenous or intraperitonealor subcutaneous injection is a preferred method of administration to aliving patient. Suitable vehicles for such injections arestraightforward. In addition, however, administration may also beeffected through the mucosal membranes by means of nasal aerosols orsuppositories. Suitable formulations for such modes of administrationare well known and typically include surfactants that facilitatecross-membrane transfer. Such surfactants are often derived fromsteroids or are cationic lipids, such asN-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethyl ammonium chloride (DOTMA) orvarious compounds such as cholesterol hemisuccinate, phosphatidylglycerols and the like.

For therapeutic applications, a therapeutically effective amount of acomposition of the invention is administered to a subject. A“therapeutically effective amount” is an amount of the therapeuticcomposition sufficient to produce a measurable response (e.g., animmunostimulatory, an anti-angiogenic response, a cytotoxic response,tumor regression, immunoinhibitory, immunosuppression, infectionreduction). Actual dosage levels of active ingredients in a therapeuticcomposition of the invention can be varied so as to administer an amountof the active compound(s) that is effective to achieve the desiredtherapeutic response for a particular subject. The selected dosage levelwill depend upon a variety of factors including the activity of thetherapeutic composition, formulation, the route of administration,combination with other drugs or treatments, tumor size and longevity,the autoimmune disease, infection, and the physical condition and priormedical history of the subject being treated. In some embodiments, aminimal dose is administered, and dose is escalated in the absence ofdose-limiting toxicity. Determination and adjustment of atherapeutically effective dose, as well as evaluation of when and how tomake such adjustments, are known to those of ordinary skill in the artof medicine. In an aspect, a typical dose contains from about 10 IU/kgto about 1,000,000 IU/kg of a cytokine described herein. In anembodiment, a typical dose contains from about 10 IU/kg to about 100IU/kg. In another embodiment, a typical dose contains about 100 IU/kg toabout 1,000 IU/kg. In still another embodiment, a typical dose containsabout 1,000 IU/kg to about 10,000 IU/kg. In yet still anotherembodiment, a typical dose contains about 10,000 IU/kg to about 100,000IU/kg. In a different embodiment, a typical dose contains about 100,000IU/kg to about 1,000,000 IU/kg. In certain embodiments, a typical dosecontains about 500,000 IU/kg to about 1,000,000 IU/kg. In otherembodiments, a typical dose contains about 100,000 IU/kg to about500,000 IU/kg. Alternatively, a typical dose contains about 50,000 IU/kgto about 100,000 IU/kg. In another embodiment, a typical dose containsabout 10,000 IU/kg to about 50,000 IU/kg. In still another embodiment, atypical dose contains about 5,000 IU/kg to about 10,000 IU/kg. In aspecific embodiment, a typical dose contains about 5,000 IU/kg to about200,000 IU/kg. In another specific embodiment, a typical dose containsabout 5,000 IU/kg to about 500,000 IU/kg. In still another specificembodiment, a typical dose contains about 50,000 IU/kg to about 500,000IU/kg. In still yet another specific embodiment, a typical dose containsabout 250,000 IU/kg to about 750,000 IU/kg.

The frequency of dosing may be once, twice, three times or more daily oronce, twice, three times or more per week or per month, as needed as toeffectively treat the symptoms or disease. In certain embodiments, thefrequency of dosing may be once, twice or three times daily. Forexample, a dose may be administered every 24 hours, every 12 hours, orevery 8 hours. In a specific embodiment, the frequency of dosing may betwice daily.

Duration of treatment could range from a single dose administered on aone-time basis to a life-long course of therapeutic treatments. Theduration of treatment can and will vary depending on the subject and thecancer or autoimmune disease or infection to be treated. For example,the duration of treatment may be for 1 day, 2 days, 3 days, 4 days, 5days, 6 days, or 7 days. Or, the duration of treatment may be for 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks. Alternatively, theduration of treatment may be for 1 month, 2 months, 3 months, 4 months,5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months,or 12 months. In still another embodiment, the duration of treatment maybe for 1 year, 2 years, 3 years, 4 years, 5 years, or greater than 5years. It is also contemplated that administration may be frequent for aperiod of time and then administration may be spaced out for a period oftime. For example, duration of treatment may be 5 days, then notreatment for 9 days, then treatment for 5 days.

The timing of administration of the treatment relative to the diseaseitself and duration of treatment will be determined by the circumstancessurrounding the case. Treatment could begin immediately, such as at thetime of diagnosis, or treatment could begin following surgery. Treatmentcould begin in a hospital or clinic itself, or at a later time afterdischarge from the hospital or after being seen in an outpatient clinic.

Although the foregoing methods appear the most convenient and mostappropriate and effective for administration of a composition of theinvention, by suitable adaptation, other effective techniques foradministration, such as intraventricular administration, transdermaladministration and oral administration may be employed provided properformulation is utilized herein.

In addition, it may be desirable to employ controlled releaseformulations using biodegradable films and matrices, or osmoticmini-pumps, or delivery systems based on dextran beads, alginate, orcollagen.

(b) Tumor

A composition of the invention may be used to treat or recognize a tumorderived from a neoplasm or a cancer. The neoplasm may be malignant orbenign, the cancer may be primary or metastatic; the neoplasm or cancermay be early stage or late stage. Non-limiting examples of neoplasms orcancers that may be treated include acute lymphoblastic leukemia, acutemyeloid leukemia, adrenocortical carcinoma, AIDS-related cancers,AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas(childhood cerebellar or cerebral), basal cell carcinoma, bile ductcancer, bladder cancer, bone cancer, brainstem glioma, brain tumors(cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, visual pathway and hypothalamic gliomas), breast cancer,bronchial adenomas/carcinoids, Burkitt lymphoma, carcinoid tumors(childhood, gastrointestinal), carcinoma of unknown primary, centralnervous system lymphoma (primary), cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, cervical cancer, childhood cancers,chronic lymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma,desmoplastic small round cell tumor, endometrial cancer, ependymoma,esophageal cancer, Ewing's sarcoma in the Ewing family of tumors,extracranial germ cell tumor (childhood), extragonadal germ cell tumor,extrahepatic bile duct cancer, eye cancers (intraocular melanoma,retinoblastoma), gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germcell tumors (childhood extracranial, extragonadal, ovarian), gestationaltrophoblastic tumor, gliomas (adult, childhood brain stem, childhoodcerebral astrocytoma, childhood visual pathway and hypothalamic),gastric carcinoid, hairy cell leukemia, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,hypothalamic and visual pathway glioma (childhood), intraocularmelanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer (renalcell cancer), laryngeal cancer, leukemias (acute lymphoblastic, acutemyeloid, chronic lymphocytic, chronic myelogenous, hairy cell), lip andoral cavity cancer, liver cancer (primary), lung cancers (non-smallcell, small cell), lymphomas (AIDS-related, Burkitt, cutaneous T-cell,Hodgkin, non-Hodgkin, primary central nervous system), macroglobulinemia(Waldenström), malignant fibrous histiocytoma of bone/osteosarcoma,medulloblastoma (childhood), melanoma, intraocular melanoma, Merkel cellcarcinoma, mesotheliomas (adult malignant, childhood), metastaticsquamous neck cancer with occult primary, mouth cancer, multipleendocrine neoplasia syndrome (childhood), multiple myeloma/plasma cellneoplasm, mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, myelogenous leukemia(chronic), myeloid leukemias (adult acute, childhood acute), multiplemyeloma, myeloproliferative disorders (chronic), nasal cavity andparanasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma,non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer,oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma ofbone, ovarian cancer, ovarian epithelial cancer (surfaceepithelial-stromal tumor), ovarian germ cell tumor, ovarian lowmalignant potential tumor, pancreatic cancer, pancreatic cancer (isletcell), paranasal sinus and nasal cavity cancer, parathyroid cancer,penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma,pineal germinoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors (childhood), pituitary adenoma, plasma cellneoplasia, pleuropulmonary blastoma, primary central nervous systemlymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidneycancer), renal pelvis and ureter transitional cell cancer,retinoblastoma, rhabdomyosarcoma (childhood), salivary gland cancer,sarcoma (Ewing family of tumors, Kaposi, soft tissue, uterine), Sézarysyndrome, skin cancers (nonmelanoma, melanoma), skin carcinoma (Merkelcell), small cell lung cancer, small intestine cancer, soft tissuesarcoma, squamous cell carcinoma, squamous neck cancer with occultprimary (metastatic), stomach cancer, supratentorial primitiveneuroectodermal tumor (childhood), T-Cell lymphoma (cutaneous),testicular cancer, throat cancer, thymoma (childhood), thymoma andthymic carcinoma, thyroid cancer, thyroid cancer (childhood),transitional cell cancer of the renal pelvis and ureter, trophoblastictumor (gestational), unknown primary site (adult, childhood), ureter andrenal pelvis transitional cell cancer, urethral cancer, uterine cancer(endometrial), uterine sarcoma, vaginal cancer, visual pathway andhypothalamic glioma (childhood), vulvar cancer, Waldenströmmacroglobulinemia, and Wilms tumor (childhood). In certain embodiments,the neoplasm or cancer may be selected from the group consisting ofmelanoma, renal cell carcinoma, lung cancer and blood cancer. As usedherein, a “blood cancer” is a cancer that affects the blood, bone marrowand lymphatic system. There are three main groups of blood cancer:leukemia, lymphoma and myeloma. The four broad classification ofleukemia are: acute lymphocytic leukemia (ALL), acute myelogenousleukemia (AML), chronic lymphocytic leukemia (CLL) and chronicmyelogenous leukemia (CML).

Lymphomas are divided into two categories: Hodgkin lymphoma andnon-Hodgkin lymphoma. Most non-Hodgkin lymphomas are B-cell lymphomas,and either grow quickly (high-grade) or slowly (low-grade). There are 14types of B-cell non-Hodgkin lymphomas. The rest are T-cell lymphomas,named after a different cancerous white blood cell, or lymphocyte.Because myeloma frequently occurs at many sites in the bone marrow, itis often referred to as multiple myeloma.

(c) Subject

A suitable subject includes a human, a livestock animal, a companionanimal, lab animal, or a zoological animal. In one embodiment, thesubject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. Inanother embodiment, the subject may be a livestock animal. Non-limitingexamples of suitable livestock animals may include pigs, cows, horses,goats, sheep, llamas and alpacas. In yet another embodiment, the subjectmay be a companion animal. Non-limiting examples of companion animalsmay include pets such as dogs, cats, rabbits, and birds. In yet anotherembodiment, the subject may be a zoological animal. As used herein,a“zoological animal” refers to an animal that may be found in a zoo.Such animals may include non-human primates, large cats, wolves, andbears. In a specific embodiment, the animal is a laboratory animal.Non-limiting examples of a laboratory animal may include rodents,canines, felines, and non-human primates. In certain embodiments, theanimal is a rodent. Non-limiting examples of rodents may include mice,rats, guinea pigs, etc. In preferred embodiments, the subject is ahuman.

TABLE A Sequences SEQ ID NO Name Sequence Source 1 R38A,CACAAACTCGCATTCAACTTCAATCTAGAAATAAATG Synthesized F42K,GCAGTGATACACATTCTACAGTAGATGTATATCTTG C125S IL2-ATGATTCTCAAATTATAACGTTTGATGGAAAAGAT OMCPATCCGTCCAACCATCCCGTTCATGATAGGTGATGAA constructATTTTCTTACCGTTTTATAAAAATGTGTTTAGTGAGTTTTTCTCTCTGTTTAGAAGAGTTCCTACAAGTACTCCATATGAAGACTTGACATATTTTTATGAATGCGACTATACAGACAATAAATCTACATTTGATCAGTTTTATCTTTATAATGGCGAAGAATATACTGTCAAAACACAGGAGGCCACTAATAAAAATATGTGGCTAACTACTTCCGAGTTTAGACTAAAAAAATGGTTCGATGGCGAAGATTGTATAATGCATCTTAGATCGTTAGTTAGAAAAATGGAGGACAGTAAACGAAACACTGGTGGTACCGGAAGTAGCGGTAGTAGTGATTACAAGGACGATGACGACAAGCACCACCATCATCATCATCACCACGGTAGCAGCGGCAGCAGTGCCCCCACCTCTAGCAGCACAAAGAAGACCCAGCTGCAACTGGAACACCTCCTGCTGGACCTGCAGATGATCCTGAACGGCATCAACAACTACAAGAACCCCAAGCTGACCGCCATGCTGACCAAAAAGTTTTACATGCCCAAGAAGGCCACCGAGCTTAAACACCTGCAATGCCTTGAGGAGGA GCTGAAGCCCTGGAGGAGGTACTGAACCTGGCCCAGAGCAAGAACTTTCATCTGAGGCCCAGGGACCTGATTAGCAACATCAACGTGATCGTGTTGGAGTTGAAGGGCAGCGAGACCACGTTCATGTGCGAGTACGCCGACGAGACGGCCACCATAGTGGAGTTTCTTAACAGGTGGAT CACCTTCTCACAGTCTATCATCAGCACCCTGACC2 R38A, HKLAFNFNLEINGSDTHSTVDVYLDDSQIITFDGKDIRPT Synthesized F42K,IPFMIGDEIFLPFYKNVFSEFFSLFRRVPTSTPYEDLTYF C125S IL2-YECDYTDNKSTFDQFYLYNGEEYTVKTQEATNKNMWL OMCPTTSEFRLKKWFDGEDCIMHLRSLVRKMEDSKRNTGGT constructGSSGSSDYKDDDDKHHHHHHHHGSSGSSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQ SIISTLT 3 Melanoma SVYDFFVWLHomo tumor sapiens associate antigen tyrosinase- related protein 2peptide 4 Highly SIINFEKL Homo immunoge sapiens nic peptide 5 WT IL2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM Homo (C125S)LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNF sapiensHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFL NRWITFCQSIISTLT 6 R38A,APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAM Synthesized F42K,LTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNF C125S IL2HLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFL NRWITFSQSIISTLT 7 OMCPHKLAFNFNLEINGSDTHSTVDVYLDDSQIITFDGKDIRPT SynthesizedIPFMIGDEIFLPFYKNVFSEFFSLFRRVPTSTPYEDLTYFYECDYTDNKSTFDQFYLYNGEEYTVKTQEATNKNMWL TTSEFRLKKWFDGEDCIMHLRSLVRKMEDSKRNT8 Linker GSSGSSDYKDDDDKHHHHHHHHGSSGSS Synthesized 9 FLAG tag DYKDDDKSynthesized 10 HA tag YPYDVPDYA Synthesized 11 Myc tag EQKLISEEDLSynthesized 12 V5 tag GKPIPNPLLGLDST Synthesized 13 OMCPbrGHKLAFNFNLEINGSDTHSTVDVYLDDSQIITFDGKDIRP Cowpox virusTIPFMIGDEIFLPFYKNVFSEFFSLFRRVPTSTPYEDLTYFYECDYTDNKSTFDQFYLYNGEEYTVKTQEATNKNMW LTTSEFRLKKWFDGEDCIMHLRSLVRKMEDSKR14 OMCPmpx HKLVHYFNLKINGSDITNTADILLDNYPIMTFDGKDIYPSI MonkeypoxAFMVGNKLFLDLYKNIFVEFFRLFRVSVSSQYEELEYYY virusSCDYTNNRPTIKQHYFYNGEEYTEIDRSKKATNKNSWLI TSGFRLQKWFDSEDCIIYLRSLVRRMEDSNK15 MICA MEPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRC HomoRDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNG sapiensKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETKEWTMPQSSRAQTLAMNVRNFL KEDAMKTKTHYHAMHADCLQELRRYLKSGVVLR16 MICB MEPHSLRYNLMVLSQDGSVQSGFLAEGHLDGQPFLRY HomoDRQKRRAKPQGQWAEDVLGAETWDTETEDLTENGQD sapiensLRRTLTHIKDQKGGLHSLQEIRVCEIHEDSSTRGSRHFYYNGELFLSQNLETQESTVPQSSRAQTLAMNVTNFWKE DAMKTKTHYRAMQADCLQKLQRYLKSGVAIR 17ULBP3 DAHSLWYNFTIIHLPRHGQQWCEVQSQVDQKNFLSYD HomoCGSDKVLSMGHLEEQLYATDAWGKQLEMLREVGQRL sapiensRLELADTELEDFTPSGPLTLQVRMSCECEADGYIRGSWQFSFDGRKFLLFDSNNRKWTVVHAGARRMKEKWEKD SGLTTFFKMVSMRDCKSWLRDFLMHRKKRLE 18RAE-1B DAHSLRCNLTIKDPTPADPLWYEAKCFVGEILILHLSNIN HomoKTMTSGDPGETANATEVKKCLTQPLKNLCQKLRNKVS sapiensNTKVDTHKTNGYPHLQVTMIYPQSQGRTPSATWEFNISDSYFFTFYTENMSWRSANDESGVIMNKWKDDGEFVK QLKFLIHECSQKMDEFLKQSKEK 19 NKG2DLTIIEMQKGDCALYAS Homo portion sapiens 20 NKG2D LTIIEMQKGECALYAS Greenportion monkey 21 NKG2D LTIIEMQKGDCAVYAS Marmoset portion 22 NKG2DLTLVEIPKGSCAVYGS Mouse portion 23 NKG2D LTLVKTPSGTCAVYGS Rat portion 24NKG2D LTLMDTQNGKCALYGS Guinea pig portion 25 NKG2D LTLVEMQNGTCIVYGSGround portion squirrel 26 NKG2D LTVVEMQSGSCAVYGS Deer mouse portion 27NKG2D LSMVEMQNGTCAVYAS Naked mole portion rat 28 NKG2D LTLVEMQRGSCAVYGSPrairie vole portion 29 NKG2D VSIVEMQGGNCAVYGS European portion shrew 30NKG2D VTVYEMQNGSCAVYGS Star-nosed portion mole 31 NKG2D LTLVEMQNGSCAVYGSChinese portion hamster 32 NKG2D LTMVDMQNGTCAVYGS Cat portion 33 OMCPASSFK Cowpox virus portion 34 DAP10 YINM Synthesized signaling motif

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Introduction to Examples 1-6

The IL-2Rα chain serves to capture IL-2 at the cell surface tofacilitate subsequent binding to the signaling part of the receptor,namely the IL-2Rβγ chains. Resting cytotoxic lymphocytes, such asnatural killer (NK) and CD8⁺ T cells, do not express appreciable IL-2Rαat the cell surface and are thus not activated by low levels of IL-2¹.IL-2Rα is expressed on this population after initial activation,however, and is required for maximum cytotoxic lymphocyte expansion².High dose IL-2 can induce the activation of all cytotoxic lymphocytesand is approved for treatment of several malignancies with anapproximately 15% partial or complete tumor response³⁻⁵. Most patientsdo not benefit from such therapy due to activation of regulatory T cell(T_(regs)) and complications such as severe blood pressure alteration,generalized capillary leak, and end organ failure due to activation ofvascular endothelium^(6,3,7). Both vascular endothelium and T_(regs)express IL-2Rα and are thus preferentially activated by IL-2 overcytotoxic lymphocytes. Lowering the IL-2 dose can ameliorate sideeffects but also decreases efficacy. Mutant forms of IL-2, such as thosewith substitutions of alanine for arginine at the 38 position (R38A)and/or lysine for phenylalanine at the 42 position (F42K), decrease theaffinity of IL-2 for IL-2Rα and thus eliminate many side effects⁹. SuchIL-2α mutants may also decrease the efficacy of immunotherapy². A formof IL-2 that could preferentially activate cytotoxic lymphocytes in theabsence of IL-2Rα reactivity would be highly advantageous for clinicalapplications.

NKG2D recognizes MHC class-I-related stress ligands expressed bymalignant or virally-transformed cells¹⁰. Of all the activatingimmunoreceptors NKG2D has the highest specificity for cytotoxiclymphocytes as it is constitutively expressed on both murine and humanNK cells as well as activated CD8⁺ T cells. Consequentially it has beenargued that tumors and virally infected cells utilize shed NKG2D ligandsas a mechanism of immune evasion^(12,13). Orthopox majorhistocompatibility complex class I-like protein, or OMCP, is an NKG2Dligand decoy shed by monkeypox and cowpox virus infected cells. It isnot expressed by small pox or vaccinia virus and thus not recognized bythose immunized with small pox vaccine. As OMCP binds to both human andmurine NKG2D with the highest affinity of any known ligand we thought itmight function as an ideal targeting vector to optimally deliver IL-2 tocytotoxic lymphocytes^(14,15). Here we describe the construction andfunction of a fusion protein designed to deliver an IL-2Rα mutant toNKG2D-expressing lymphocytes¹⁵. We demonstrate that this constructovercomes decreased efficacy associated with mutations in the IL-2Rαbinding region while retaining a favorable safety profile. Systemicadministration of this fusion protein improves immunotherapy againstboth solid and liquid tumors. Targeted delivery of IL-2 can thus besafely used to maximally activate NKG2D-expressing lymphocytes, such asNK cells, to optimize immunotherapy without systemic side effects.

Example 1. NKG2D-Targeted Delivery of an IL-2 Mutant PreferentiallyActivates Cytotoxic Lymphocytes In Vitro

To overcome the preferential activation of IL-2Ra-expressing cells, wedesigned an IL-2 fusion protein that would target cytotoxic lymphocytesdirectly via the NKG2D receptor. This fusion protein combines the highaffinity NKG2D ligand OMCP with an IL-2 mutated to reduce IL-2Rareactivity. Our construct, termed OMCP-mutIL-2, consists of the 152residue OMCP protein fused to the N-terminus of the 133 amino acidR38A/F42K mutant form of human IL-2 (mutIL-2) via a flexible 30 residuelinker (FIG. 1A-B). The construct was first assessed for its in vitrobinding ability. Binding of fluorescently labeled construct was testedin vitro at 37° C. in bulk splenocytes. FIG. 17 shows that the constructappears to only bind to NK cells which express NKG2D. The construct doesnot show binding to CD4+CD3+ T cells, CD8+CD3+ T cells, CD11C+CD11b−DCs, CD11c−CD11b+ Macs or CD19+CD3− B cells.

We have previously demonstrated strain-specific differences in murine NKcell cytotoxicity and lung cancer immunosurveillance¹⁸ (and Example 7).Therefore, we set out to examine the efficacy of OMCP-mut-IL2 inactivation of NK cells from two different strains of mice, namely A/Jand B6 with poor and robust NK function, respectively. Compared towild-type IL-2 (wtIL-2) or mutIL-2, OMCP-mutIL-2 strongly upregulatedCD69 on NK cells of both strains after 36-hour co-culture with 100IUe/ml of cytokine (FIG. 1C, left; FIG. 6, left two graphs)^(16,17). Athigh concentrations a similar increase in CD69 expression was observedwith OMCP-mut-IL-2, wtIL-2 or mutIL-2 (FIG. 6). Activation of CD4⁺Foxp3⁺T_(regs), as measured by upregulation of ICOS, was evident with wtIL-2only, but not with mutIL-2 nor OMCP-mutIL-2 (FIG. 1C-D). CD8⁺ andCD4⁺Foxp3⁻ effector T cells, on the other hand, demonstrated noupregulation of CD69 after 36 hours, even at highest doses of cytokines(FIGS. 1C-D and data not shown). Longer exposure over a period of fivedays led to proliferation of both NK and CD8⁺ T cells exposed to wtIL-2and OMCP-mutIL-2 (FIGS. 1E-F). Importantly, OMCP-mutIL-2 activated CD8⁺T cells and NK cells equivalently to mutIL-2 in NKG2D^(−/−) splenocytes,indicating that the increased activation was due to the effect of OMCPtargeting upon NKG2D-bearing cells (FIG. 1F; FIG. 6, right two graphs).Only incubation with wtIL-2 led to CD4⁺Foxp3⁺ T_(regs) and CD4⁺Foxp3⁻effector cell proliferation (FIG. 1E-F). Thus exposure to OMCP-mutIL-2results in preferential NK activation that is superior or equivalent towtIL-2 in a dose-dependent manner. CD8⁺ T cells can also be activatedbut require prolonged exposure to higher doses of OMCP-mutIL-2.

Example 2. Low-Dose Cytokine Therapy Offers a Favorable Safety Profile

Dose-dependent toxicity can limit cytokine administration in vivo. Tomodel human immunotherapy protocols we next treated A/J mice with wtIL-2given as ten doses over a five day cycle¹⁸. While A/J mice tolerated750,000 IUe of wtIL-2, significant mortality was evident at higher doses(FIG. 2A-B). Even after a 750,000 IUe dose mice demonstrated extremedistress, weight loss, decreased food consumption, ascites and hepaticinflammation (FIG. 2A-E; FIG. 7A-C). These side-effects mirror thecapillary leak and distress associated with high dose IL-2 therapy inhumans⁷. Treatment with anti-Asialo-GM1 ameliorated mortality, but notweight loss, induced by high dose wtIL-2 (1,500,000 IUe) in A/J mice,confirming that side effects of such therapy can occur independent of NKcells (FIG. 2F-K). Unlike the case for wtIL-2 no animal death wasevident after 1,500,000 IUe of OMCP-mutIL-2 or mutIL-2 in the presenceor absence of NK cells. Animal weight loss after 1,500,000 IUe ofOMCP-mutIL-2 occurred only in NK-sufficient mice suggesting thattoxicity of our construct was solely due to immunoactivation (FIG.2F-K). A regimen of 200,000 IUe was well tolerated in A/J mice withminimal weight loss, distress, or organ inflammation for all cytokines(FIG. 2L-O). Capillary leak, however, was still evident by accumulationof pleural effusion and ascites after wtIL-2, but not OMCP-mutIL-2 ormutIL-2, at this dose. B6 mice were able to tolerate higher doses ofwtIL-2 but still suffered significant morbidity over 750,000 IUe (FIG.7D).

Example 3. OMCP-mutIL-2 Preferentially Expands and Activates NK Cells InVivo Compared to wtIL-2 or mutIL-2

To evaluate immunologic changes associated with cytokine treatment, A/Jmice received 200,000 IUe of cytokine or construct given as ten equaldoses over five days. Splenic lymphocytes were evaluated flowcytometrically on day six. Both wtIL-2 and OMCP-mutIL-2 increasedlymphocyte content and splenic size over saline-treated controls (FIG.3A-B). OMCP-mutIL-2 led to a substantial expansion and activation of NKcells measured by cellularity and surface KLRG1 levels (FIG. 3C). InOMCP-mutIL-2 treated mice NK cells comprised close to half of allsplenic lymphocytes, paralleling or even surpassing the total lymphocytecounts of saline or mutIL-2-treated mice (FIG. 3A vs. FIG. 3C). NKexpansion by 200,000 IUe of OMCP-mutIL-2 was superior to near toxicdoses of wtIL-2 (750,000 IU), high dose mutIL-2 (3,500,000 IUe), orwtIL-2 complexed to anti-IL-2 antibody (clone MAB602)¹⁹ (FIG. 3C). Infact, the majority of mice could not tolerate the full 200,000 IUe ofwtIL-2/anti-IL-2 antibody and injections had to be terminated at 160,000or 180,000 IUe with requisite animal sacrifice due to animal distressand rapid weight loss (FIG. 8A). WtIL-2 led to a significant expansionof CD4⁺Foxp3⁺ T_(regs), specifically the ICOS⁺ Subset® in A/J mice evenwhen complexed to anti-IL-2 antibodies (FIG. 3D). Importantly theNK/T_(reg) ratio, which has been described as a predictive factor forsuccess of immunotherapy²⁰, was dramatically increased in OMCP-mutIL-2treated mice compared to all other treatment conditions (FIG. 3E).Superior expansion of NK cells by OMCP-mutIL-2 was even possible atdoses 2-fold lower than wtIL-2 (FIG. 8B). However, targeting NKG2D witha ˜500-fold lower affinity NKG2D ligand, ULBP3, ameliorated efficacy ofthe fusion construct for expansion but still offered superior NKactivation compared to mutIL-2 alone (FIG. 8B). No statisticallysignificant increase in CD4⁺Foxp3⁻ or CD8⁺ T lymphocytes was evidentafter wtIL-2 or OMCP-mutIL-2 treatment, although a trend for CD8⁺ T cellexpansion was evident (FIG. 8C-D). Such data is consistent with theprevalence of naïve T lymphocytes, expressing low levels of IL-2receptors and NKG2D in specific pathogen-free mice.

Unlike the A/J strain little immunoactivation of lymphocytes was evidentin B6 mice treated with 200,000 IUe of wtIL-2 (data not shown). Athigher doses of 750,000 IUe OMCP-mutIL-2 expanded NK cells more robustlythan wtIL-2 in this strain (FIG. 3F-H). IL-2/anti-IL-2 antibodycomplexes prevented T_(reg) expansion but, similar to the NJ strain,such treatment had toxicity and the majority of B6 mice could nottolerate the full 750,000 IUe dose (FIG. 3I). OMCP-mutIL-2, however, waswell tolerated at this dose and led to a high NK/T_(reg) ratio (FIG.3J). No expansion of NK cells was evident in OMCP-mutIL-2 treated B6NKG2D^(−/−) mutants, confirming the requirement for NKG2D in thefunction of our construct (data not shown). No statistically significantexpansion of B6 CD8⁺ or CD4⁺Foxp3⁻ T cells was evident in any treatmentgroup although a trend for CD8⁺ T cell expansion was evident afterwtIL-2 administration (FIG. 8F-G). Identical data was obtained for lungresident lymphocytes in both the A/J and B6 strains (data not shown).

Example 4. OMCP-mutIL-2 Preferentially Expands and Activates NK Cells inHuman Peripheral Blood Lymphocytes Compared to wtIL-2 or mutIL-2

To demonstrate the effectiveness of OMCP-mutIL-2 in human lymphocytes,human peripheral blood lymphocytes were co-cultured for 36 hours in 100IUe of either wild-type IL2, R38A/F42K mutant form of IL-2 orOMCP-mutant IL-2.

NK cells: The cells were flow cytometrically analyzed and relativeprevalence of CD56+CD3− NK cells compared between conditions. Arelatively higher proportion of NK cells was evident in the OMCP-mutantIL-2 group (FIG. 31A). Perforin levels were higher in OMCP-mutant IL-2treated NK cells (red) compared to saline (black), IL-2 (blue) or mutantIL-2 (green) treated ones (FIG. 31B).

CD8+ T cells: Similar to NK cells, higher intracellular levels ofperforin were evident in CD8+ T cells treated with OMCP-mutant IL-2compared to other conditions (FIG. 31C).

Tregs: When gating on CD4+Foxp3+CD45RA− T cells a relatively higherproportion of activated CD25+CD127− regulatory T cells was evident inIL-2 treated peripheral blood lymphocyte cultures compared to otherconditions (FIG. 31D). Taken together this data suggests thatOMCP-mutIL-2 preferentially expands and activates NK cells and CD8+cells in human peripheral blood lymphocytes compared to wtIL-2 ormutIL-2. Importantly, OMCP-mutIL-2 does not activated regulatory T cellssignificantly relative to IL2.

Example 5. Treatment with OMCP-mutIL-2 Offers Superior ImmunologicControl of Malignancies In Vivo

Unlike T lymphocytes, which require prior antigen encounter for optimalantigen-specific tumor cytotoxicity, NK cells can mediate naturalcytotoxicity without prior sensitization. NK cells also form the primarybarrier for expansion of select malignancies, such as lymphoma and lungcancer^(16,17,21,22). Treatment of AJ mice with OMCP-mutIL-2, comparedto wtIL-2 or mutIL-2, led to enhanced in vivo clearance and in vitrolysis of YAC-1 cells by bulk splenocytes (FIG. 4A, FIG. 9A-B). Decreasedgrowth of the highly aggressive Lewis Lung Carcinoma (LLC) cell line wasevident in B6 mice after 750,000 IUe of OMCP-mutIL-2 compared to wtIL-2or mutIL-2. Increased cytotoxicity was evident in OMCP-mutIL-2 treatedsplenocytes for the LLC cell line as well (FIG. 4B-C; FIG. 9A-C).Enhanced immunotherapy was lost in NKG2D^(−/−) mice or following NKdepletion (FIG. 4D-E). In the absence of host NKG2D mutIL-2 actuallyincreased the rate of LLC growth. Thus OMCP-mediated targeting ofmutIL-2 offers a safer and more efficacious form of immunotherapy forboth solid and liquid tumors in various strains of mice.

Example 6. Impact of NKG2D Targeting on IL-2 Signaling

Antibody-IL-2 conjugates, or IL-2/anti-IL-2 antibody complexesdemonstrate improved biologic activity over purified cytokine byextending the duration of serum half-life^(23,24). To investigatewhether linking IL-2 to OMCP increased serum half-life, we injected500,000 IUe of fluorescently-labeled wtIL-2, mutIL-2 or OMCP-mutIL-2into A/J and B6 mice and monitored serum clearance by serial blooddraws. While OMCP-mutIL-2 had a slightly higher serum concentration atearly time points, all constructs were undetectable in the blood onehour post-injection (FIG. 5A-B). This is significantly shorter than thedescribed 11-14 hour serum half-life of antibody-IL-2 conjugates²³.Interestingly, despite the injection of identical amount of cytokine,lower cytokine levels were detected in B6 mice compared to A/J mice atall time points. Such data points to strain-specific differences inclearance of IL-2 and may explain why B6 mice are able to both tolerateand require higher doses of cytokine for NK expansion. Nevertheless,based on this data it is unlikely that prolonged circulation ofconstruct was responsible for the increased activity of OMCP-mutIL-2over wtIL-2.

We next considered the possibility that the superiority of OMCP-mutIL-2was the result of signaling through NKG2D as antibody-mediatedcrosslinking of this receptor can activate NK cells (FIG. 10A)²⁵. Whilethe addition of purified OMCP to mutIL-2 did not augment NK activationor expansion in vitro or in vivo (data not shown) we would not expect amonomeric ligand to crosslink NKG2D. We thus directly compared NK cellactivation in the presence of 1000 IUe of OMCP-mutIL-2, mutIL-2 andmutIL-2 combined with equimolar concentration of pentamerized OMCP. Noincrease in NK activation, as measured by CD69 upregulation ordegranulation, was evident in the presence of pentamerized OMCP (FIG.5C, FIG. 10B). This suggests that NKG2D crosslinking is not responsiblefor augmented NK cell activation by OMCP-mutIL-2 at physiologicconcentrations.

To evaluate IL-2 signaling we next quantitated STAT5 phosphorylationafter a 15 minute cytokine stimulation of freshly isolated NK cells invitro. Lower levels of STAT5 phosphorylation were evident in A/Jcompared to B6 NK cells at all concentrations tested (FIG. 5D-E)suggesting that lymphocyte dysfunction of A/J mice may at leastpartially be the result of inefficient IL-2 signal transduction.Surprisingly, for both B6 and A/J NK cells wtIL-2 and OMCP-mutIL-2demonstrated an identical dose-dependent pattern of STAT5phosphorylation (FIG. 5D-E). In the absence of NKG2D reactivityOMCP-mutIL-2 failed to increase STAT5 phosphorylation over mutIL-2alone. Taken together these data suggested that IL-2a reactivity isimportant for peak IL-2 signaling in resting NK cells, and thatNKG2D-binding may effectively substitute for IL-2Ra-binding inIL-2-mediated signal transduction. Such data, however, failed to explainthe superior NK activation by OMCP-mutIL-2 in vivo or in bulk splenocytecultures (FIG. 1C-D, FIG. 3).

IL-2 signaling results in the internalization of IL-2/IL-2R, withsubsequent degradation of IL-2 and IL-2Rβγ. The binding of OMCP-mutIL-2to both the IL-2 receptor and NKG2D could thus lead to alteredinternalization and enhanced NK cell activation by prolonging IL-2signaling. To test this we stimulated freshly isolated NK cells for 15minutes, replaced the culture media with cytokine free media, andmonitored STAT5 phosphorylation for four hours. Identical decay ofphospho-STAT5 was evident for both wtIL-2 and OMCP-mutIL-2 (FIG. 5F-G).Thus altering duration of IL-2 signaling is not responsible for superiorNK activation by OMCP-mutIL-2.

We next considered the possibility that superior NK activation byOMCP-mutIL-2 may be the result of altered cytokine interaction withcompeting stromal cells (FIG. 5H). Indeed, in the presence of othersplenocytes OMCP-mutIL-2 demonstrated a dose-dependent enhancement in NKSTAT5 phosphorylation over wtIL-2 (FIG. 5I). We next explored theinterplay between IL-2Ra expression by stromal cells and NKG2Dexpression by NK cells on IL-2 signal transduction. To accomplish thiswe isolated splenic NK cells from either wild-type or NKG2D^(−/−) B6mice and combined them with wild-type splenocytes depleted of NK cells.Cultures were recombined in a 1:20 NK:splenocyte ratio, resembling theproportion normally present in resting wild-type B6 mice. For somecultures NK cell depleted splenocytes were treated with saturatingconcentrations of IL-2Ra-blocking antibody (clone 3C7) prior torecombining with wild-type NK cells. The cultures were then stimulatedwith 1000 IUe of either wtIL-2 or OMCP-mutIL-2 for 15 minutes. STAT5phosphorylation was identical in NKG2D^(−/−) or wild type NK cells inthe presence of wtIL-2 (FIG. 5J, left two columns). Wild-type NK cellscultured with OMCP-mutIL-2 demonstrated superior STAT5 phosphorylationto cultures with wtIL-2. Little STAT5 phosphorylation was evident inNKG2D^(−/−) NK cells cultured with OMCP-mutIL-2 (FIG. 5J, right twocolumns). In the presence of IL-2Ra-blockade of competing splenocytestromal cells, NK cell STAT5 phosphorylation by wtIL-2 increased tolevels comparable to OMCP-mutIL-2 (FIG. 5K). Taken together these datademonstrate that IL-2-Rα expression by “competing” stromal cells limitsNK cell activation by wtIL-2 and this competition can be eliminated bythe NKG2D-targeted, IL-2Rα-binding impaired OMCP-mutIL-2 construct.

Discussion for Examples 1-6

While IL-2 therapy initially showed great promise, it has been limitedby activation of T_(regs) and toxic side effects associated withactivation of vascular endothelium. Several strategies have beenproposed to preferentially activate cytotoxic lymphocytes. One strategyhas been to create mutants with increased affinity for IL-2R3 to removethe preference for IL-2Rα^(26,27). Importantly, these IL-2 mutantsretain wild type binding for IL-2Rα, and would therefore still berecognized by T_(reg) cells and vascular endothelium. Our results alsosuggest that competition with IL2-Rα⁺-expressing cells limitsbioavailability of wtIL-2 to cytotoxic lymphocytes.

Another promising therapy involves anti-IL2 antibodies that stericallyinhibit wtIL-2 binding to IL-2Rα^(1,28,29). Such treatment can extendserum half-life²⁴ due to the Fc region of the antibody and potentiallydue to reduced competition for wtIL-2 from IL-2Rα-expressing cells.Antibody-IL-2 fusion proteins have also been designed to target IL-2 tospecific tumor antigens^(30,31). While offering the potential forpersonalized therapy such antibody-mediated delivery of IL-2 to thetumor depends on the expression of a known tumor associated antigen, asituation that often does not exist. This approach could potentially befurther limited by tumor-mediated alteration of the targeted antigen.

Finally, IL-2 mutants with reduced affinity for IL-2Ra have been testedextensively. Compared to wtIL-2 these mutants can be administered insupratherapeutic doses without IL-2Rα-mediated capillary leak orsystemic toxicity³². While these mutants have excellent safety profiles,they activate cytotoxic lymphocytes poorly (FIG. 5C-E)³³. Our approachcombines several of the concepts above to target a safe form of IL-2directly to cytotoxic lymphocytes, instead of tumors. This isaccomplished by replacing the normal targeting of IL-2 to IL-2Rα withNKG2D. The combination of an IL-2Rα-deficient IL-2 fused to a highaffinity NKG2D-ligand improves upon previous strategies by specificallyexpanding NK cells without any apparent activation of T_(regs) orcapillary leak. These findings offer the promise of a potentially safeand highly efficacious form of IL-2.

One limitation in translating results from inbred lab animals to humansis the natural diversity in cytokine reactivity and environmentallydependent threshold for lymphocyte activation. Previous studies havedemonstrated a correlation between ex vivo killing of tumor cells andenhanced long-term cancer immunity³⁴. Therefore, any potential therapyneeds to account for a population that has differential levels ofcytotoxic lymphocyte activity. We have thus attempted to model thisnatural variation by using two strains of mice known to be highlyresistant (B6) or susceptible (A/J) to carcinogenesis. For example, NKcells from B6 mice, are activated by wtIL-2 and extreme doses ofmutIL-2. In contrast, IL-2/anti-IL-2 antibody complexes resulted inexpansion of NK cells in A/J but not in B6 mice. Such variationshighlight the limitations of translating results derived from a singlestrain of mice to immunologically diverse humans. Importantly, theOMCP-mutIL-2 construct was able to expand NK cells in both strains ofmice, indicating that this therapy could be efficacious in populationswith diverse NK function and cytokine reactivity.

Since OMCP has been described as an evolutionary antagonist of NKG2D³⁵blockade of this immunoreceptor at the time of tumor therapy may beconstrued as counterproductive. Nevertheless, natural cytotoxicity andtumor clearance was augmented in OMCP-mutIL-2-treated mice even in thepresence of established tumors. This suggests minimal or transient NKG2Dreceptor occupancy and preservation of function. Alternatively recentreports have demonstrated that shed NKG2D ligands may actually promotetumor immunity through reversal of NK desensitization imposed by chronicagonistic engagement³⁶. While we did not detect NK activation orexpansion by monomeric or even pentameric OMCP, it is possible thatwithin the tumor bed such competitive antagonism plays a paradoxicalrole in NK activation. In addition, IL-2 may upregulate receptorsnecessary for NK migration and tumor infiltration. It is thus possiblethat anti-tumor immunity mediated by OMCP-mutIL-2 may depend on NK cellslocated outside the tumor bed and not subject to local tumor-specifictolerance or anergy. Furthermore, OMCP maybe the ideal “targetingvector” due to its high affinity and long half-life of binding to humanNKG2D.

While NK cells from two separate strains of mice were activated byOMCP-mutIL-2 we did not detect global expansion of activation of CD8⁺ Tcells by our construct. This is most likely due to the fact that NKG2Dis expressed only on select subsets of CD8⁺ T cells, namely memory oractivated cytotoxic lymphocytes. Based on the paucity of this cellpopulation in mice raised in specific pathogen-free environment,OMCP-mutIL-2-mediated activation was limited in our system to NK cells.To this end we focused on immunotherapy for lung cancer and lymphoma,whose growth is regulated primarily by NK cells^(16,17,22,37).Nevertheless OMCP-mutIL-2 was able to expand CD8⁺ T cells whenadministered in high concentrations in vitro (FIG. 1E-F).

Thus, it may be possible that NKG2D-targeted delivery ofimmunostimulatory cytokines may lead to the expansion and/or activationof antigen-specific CD8⁺ memory cells for long-term tumor immunity undernormal immunologic conditions.

Methods for Examples 1-6

Cytokine and Construct Generation: The sequences encoding human IL-2(1-133; C125S) and mutant IL2 (1-133; R38A, F42K, C125S) were clonedinto the pFM1.2R³⁸ with an N-terminal FLAG/hexahistidine tag. Thechimeric OMCP-mutIL-2 molecule comprises the full-length OMCP (1-152)coding sequence cloned in frame with a C-terminal FLAG/hexahistidinetag-mutant IL-2 (1-133; R38A, F42k, C125S) cloned into the pFM1.2Rvector. Proteins were expressed by transient transfection into HEK293F(Life_Technologies). Supernatant was recovered at 72 h and 144 hpost-transfection. Supernatants were supplemented with 5 mM imidazoleand 0.02% sodium azide and purified by nickel-nitrilotriacetic acid(Ni-NTA) chromatography (Qiagen). Purified proteins were bufferexchanged into saline and flash frozen in liquid nitrogen. Equivalent invitro and in vivo activity was documented for wild-type IL-2 generatedin house and Teceleukin (Tecin™) available from the NCI repository(Frederick National Laboratory for Cancer Research). Thus for someexperiments these two preparations of IL-2 were used interchangeably.

Wild-type IL-2 has a specific activity of 15×10⁶ IU/mg³⁹. Thus, based onthe molecular weight of 15.5 kDa a 4.4 μM solution is equivalent to 1000IU/μl. Based on this calculation all cytokines and construct wereadministered on a molar basis with 1 μl of 4.4 μM solution defined as1000 IU equivalents (IUe from here on). Such a system allows forequimolar comparison between IL-2, mutIL-2 and OMCP-mutIL-2 despitedifference in molecular weight.

Animals: A/J (8-12 weeks) and C57BL/6J (6-9 weeks) strains of mice werepurchased from the Jackson Laboratory (Bar Harbor, Me.). NKG2D^(−/−)mice on the B6 background were kindly provided by Wayne Yokoyama andbred in house (Howard Hughes Institute of Medicine at WashingtonUniversity in St. Louis). Animals were housed in a barrier facility inair-filtered cages and allowed free access to food and water. For someexperiments A/J mice were treated with depleting concentrations ofanti-Asialo-GM1 (50 μl day −2; 25 μl day −1) or control rabbit IgG (WakoChemical Company). Animal procedures were approved by the animal studiescommittee of Washington University School of Medicine, St. Louis, Mo.

Tissue harvest and in vitro cultures: Single cell suspension ofsplenocytes were obtained by crushing whole spleens through 70 μm cellstrainers prior to RBC lysis by ACK buffer (Lonza, Walkersville, Md.)and re-filtration through a 40 μm filter. Lungs were digested for 90minutes at 37° C. in 1 mg/ml collagenase II (Fisher Scientific), and 5U/ml DNase I (Sigma-Aldridge) prior to processing in an identicalfashion to spleens.

For in vitro cultures splenocytes from either A/J, B6, or NKG2D^(−/−)mice were extracted in a sterile fashion and seeded in 12-well plates incomplete media (RPMI 1640 supplemented with 10% FBS, 100 U/ml Penicillinand Streptomycin, 2 mM L-glutamine and 50 μM 2-Mercaptoethanol) at 5million cells per ml per well. The cells were treated with increasingdoses of human recombinant IL-2, mutIL-2, OMCP-mutIL-2, or OMCP for 36hours as described in the manuscript. For some experiments bulksplenocytes were labeled with CFSE and cultured in 1000 IUe/ml ofcytokine for 5 days prior to flow cytometric analysis. For NK isolationexperiments bulk splenocytes were processed using either the NK cellisolation kit II or CD49b (DX5) positive magnetic bead selection (bothfrom Miltenyi Biotech). For STAT5 phosphorylation experiments, isolatedNK cells were stimulated in increasing concentrations of IL-2 orconstruct at 100,000 cells/500 μl for 15 minutes. For experimentsevaluating the interaction of NK cells with splenic stroma, DX5positively selected NK cells were labeled with CFSE (for identificationafter fixation and permeabilization) and recombined with NK depletedstromal cells. As described in the manuscript, for some studiesNKG2D^(−/−) NK cells were combined with wild-type splenocyte stromalcells. For other experiments, NK-depleted splenocytes from wild-type B6mice were treated with saturating concentrations of anti-IL-2α blockingantibody (clone 3C7) or isotype control (both from Biolegend) prior torecombining with NK cells. For such competitive STAT5 phosphorylationexperiments 100,000 cells were resuspended into 2 μl complete mediacontaining 1,000 IU/ml of either wtIL2, mutIL-2 or OMCP-mut-IL-2(freshly prepared and pre-warmed). The cells were then incubated at 37°C. for 15 minutes

Flow Cytometry: All flow cytometric analysis was performed usingsaturating concentrations of fluorochrome-conjugated antibodies at 4° C.in FACS buffer consisting of PBS with 2% FBS and 0.4% EDTA. Allantibodies were anti-mouse and purchased from BD Bioscience oreBioscience and consisted of anti-CD4 (clones GK1.5 or RM4-5), anti-CD8(clone 53-6.7), anti-CD278 (ICOS) (clone: 7E.17G9), anti-CD25 (clonePC61), anti-KLRG1 (clone 2F1), CD49b (Integrin alpha 2) (clone DX5),anti-CD3e (clone 1452C11), anti-CD45 (clone 30-F11), anti-CD69 PE (cloneH1.2F3), anti-GITR (clone DTA-1), anti-Foxp3 (clone: FJK-16s) andAnti-Stat5 (clone 47/Stat5; pY694). Antibodies were conjugated to eitherFITC, PE, PerCP-CyTM5.5, PE-Cyanine7, APC, APC-eFluor®780, eFluor® 450,or Alexa Fluor® 647.

Phospho-STAT5 evaluation was performed by paraformaldehyde fixation,methanol permeabilization and staining with AlexaFluor488-conjugatedAnti-Stat5 (pY694) (BD Pharmingen; clone 612599). To accomplish thisisolated NK cells or NK cells combined with NK-depleted splenocytestromal cells were fixed in 2% paraformaldehyde (PFA) at 37° C. for 10minutes after IL-2 stimulation for 15 minutes. The cells were thenwashed once with ice-cold PBS and permeabilized by adding 0.5 ml/tube of90% Methanol on ice for 1 hour. The cells were washed once with ice-coldPBS (to remove methanol), and stained for 1 hour with anti-Stat5 (pY694)antibody at room temperature followed by one wash in PBS/0.5% fetal calfserum.

In Vitro Cytotoxicity: ⁵¹Chromium release was conducted by incubatingthe target cells with 100 mCi sodium⁵¹chromate (PerkinElmer) for 1 hour.Bulk splenocytes were used as effector cells and incubated with targetsat defined effector:target ratios for 4 hours at 37° C. in round bottom96 well plates. Specific lysis was expressed as (experimentalrelease-spontaneous release)/(maximum release-spontaneous release)×100%with 0% specific lysis as lowest expressed value.

In vivo cytokine injections: For select experiment, the mice receivedintraperitoneal injections of cytokines in 200 μl volume given as tenequal doses given twice a day over a period of five days. As describedabove all cytokines were normalized to IUe on a molar basis. For selectexperiments, the mice were then sacrificed on day 6 and organs werefixed in 10% buffered formalin for histological analyses. For otherexperiments splenocyte and lung lymphocyte populations were analyzedflow cytometrically. For all the in vivo cytokine treatment experiments,animals were weighed (daily or every other day) and expressed as %change from start of cytokine therapy.

For evaluation of serum concentration wtIL-2, mutIL-2 or OMCP-mutIL-2were labeled with Alexa Fluor® 647 (LifeTechnologies Inc.) according tomanufacturer instructions. Serum was collected at times specified andconcentration of cytokine determined fluoroscopically according to astandard curve.

In vivo tumor studies: Lewis lung carcinoma (LLC) cells weresubcutaneously injected into B6 or B6 NKG2D^(−/−) mice at 1×10⁵ cellsper mouse in 100 μl of sterile saline. Once visible tumors were evident,day 5 post-injection, a five day course of cytokine treatment wasstarted as described above. Measurement of cross sectional tumordiameter was performed using calipers and tumor volume estimated as4/3πr³. The mice were sacrificed on day 24 post injection or once theyreached a maximal tumor diameter of 20 mm. For NK cell depletion, micewere treated with anti-NK1.1 antibody (clone PK136) or mouse IgG isotypecontrol (both from BioXcell) at 500 μg day −2, 250 μg day −1 and 250 μgweekly for the duration of the experiment. For lymphoma clearanceexperiments A/J mice were treated with ten doses of cytokine over aperiod of five days as described above and on day #6 injectedintravenously with YAC-1 cells that were labeled with CFSE at 5×10⁶cells/mouse. Mice were sacrificed 4 hours later, lungs were digested andviability of YAC-1 determined by forward and side scatter analysis ofCFSE⁺ cells.

Statistics Comparison of splenic and lung-resident lymphocytes betweenvarious cytokine treatment conditions was performed by unpaired T-testwith Welch's correction to account for unequal variance or unequalsample size. Tumor growth between different cytokine conditions wascompared by multiple unpaired-T tests performed between variousconditions at various time points using the Sidak-Bonferroni correction.Fold change in STAT5 phosphorylation was evaluated by unpaired T-testwith Welch's correction in a similar fashion.

REFERENCES FOR EXAMPLES 1-6

-   1. Spangler, J. B. et al. Antibodies to Interleukin-2 Elicit    Selective T Cell Subset Potentiation through Distinct Conformational    Mechanisms. Immunity 42, 815-825 (2015).-   2. French, A. R. et al. DAP12 signaling directly augments    proproliferative cytokine stimulation of NK cells during viral    infections. J Immunol 177, 49814990 (2006).-   3. Rosenberg, S. A. et al. Experience with the use of high-dose    interleukin-2 in the treatment of 652 cancer patients. Annals of    surgery 210, 474-484; discussion 484-475 (1989).-   4. Rosenberg, S. A. IL-2: the first effective immunotherapy for    human cancer. J Immunol 192, 5451-5458 (2014).-   5. Atkins, M. B. et al. High-dose recombinant interleukin 2 therapy    for patients with metastatic melanoma: analysis of 270 patients    treated between 1985 and 1993. J Clin Oncol 17, 2105-2116 (1999).-   6. Sim, G. C. et al. IL-2 therapy promotes suppressive ICOS+ Treg    expansion in melanoma patients. J Clin Invest 124, 99-110 (2014).-   7. Kolitz, J. E. et al. Recombinant interleukin-2 in patients aged    younger than 60 years with acute myeloid leukemia in first complete    remission: results from Cancer and Leukemia Group B 19808. Cancer    120, 1010-1017 (2014).-   8. Krieg, C., Letoumeau, S., Pantaleo, G. & Boyman, O. Improved IL-2    immunotherapy by selective stimulation of IL-2 receptors on    lymphocytes and endothelial cells. Proc Natl Acad Sci USA 107,    11906-11911 (2010).-   9. Heaton, K. M., Ju, G. & Grimm, E. A. Human interleukin 2    analogues that preferentially bind the intermediate-affinity    interleukin 2 receptor lead to reduced secondary cytokine secretion:    implications for the use of these interleukin 2 analogues in cancer    immunotherapy. Cancer Res 53, 2597-2602 (1993).-   10. Ullrich, E., Koch, J., Cerwenka, A. & Steinle, A. New prospects    on the NKG2D/NKG2DL system for oncology. Oncoimmunology 2, e26097    (2013).-   11. Raulet, D. H. Roles of the NKG2D immunoreceptor and its ligands.    Nat Rev Immunol 3, 781-790 (2003).-   12. Raulet, D. H., Gasser, S., Gowen, B. G., Deng, W. & Jung, H.    Regulation of ligands for the NKG2D activating receptor. Annu Rev    Immunol 31, 413-441 (2013).-   13. Giuliani, E., Vassena, L., Cerboni, C. & Doria, M. Release of    Soluble Ligands for the Activating NKG2D Receptor: One More Immune    Evasion Strategy Evolved by HIV-1 ? Current drug targets (2015).-   14. Campbell, J. A., Trossman, D. S., Yokoyama, W. M. &    Carayannopoulos, L. N. Zoonotic orthopoxviruses encode a    high-affinity antagonist of NKG2D. J Exp Med 204, 1311-1317 (2007).-   15. Lazear, E., Peterson, L. W., Nelson, C. A. & Fremont, D. H.    Crystal structure of the cowpox virus-encoded NKG2D ligand OMCP. J    Virol 87, 840-850 (2013).-   16. Kreisel, D. et al. Strain-specific variation in murine natural    killer gene complex contributes to differences in immunosurveillance    for urethane-induced lung cancer. Cancer Res 72, 4311-4317 (2012).-   17. Frese-Schaper, M. et al. Influence of natural killer cells and    perforinmediated cytolysis on the development of chemically induced    lung cancer in A/J mice. Cancer ImmunolImmunother 63, 571-580    (2014).-   18. Dandamudi, U. B. et al. A phase II study of bevacizumab and    high-dose interleukin-2 in patients with metastatic renal cell    carcinoma: a Cytokine Working Group (CWG) study. J Immunother 36,    490-495 (2013).-   19. Boyman, O., Kovar, M., Rubinstein, M. P., Surh, C. D. &    Sprent, J. Selective stimulation of T cell subsets with    antibody-cytokine immune complexes. Science 311, 1924-1927 (2006).-   20. Smyth, M. J. et al. CD4+CD25+ T regulatory cells suppress NK    cell-mediated immunotherapy of cancer. J Immunol 176, 1582-1587    (2006).-   21. Chang, S. et al. Unique pulmonary antigen presentation may call    for an alternative approach toward lung cancer immunotherapy.    Oncoimmunology 2, e23563 (2013).-   22. Plonquet, A. et al. Peripheral blood natural killer cell count    is associated with clinical outcome in patients with aalPI 2-3    diffuse large B-cell lymphoma. Annals of oncology: official journal    of the European Society for Medical Oncology/ESMO 18, 1209-1215    (2007).-   23. Tzeng, A., Kwan, B. H., Opel, C. F., Navaratna, T. &    Wttrup, K. D. Antigen specificity can be irrelevant to    immunocytokine efficacy and biodistribution. Proc Natl Acad Sci USA    112, 3320-3325 (2015).-   24. Letoumeau, S. et al. IL-2/anti-IL-2 antibody complexes show    strong biological activity by avoiding interaction with IL-2    receptor alpha subunit CD25. Proc Natl Acad Sci USA 107, 2171-2176    (2010).-   25. Ho, E. L. et al. Costimulation of multiple NK cell activation    receptors by NKG2D. J Immunol 169, 3667-3675 (2002).-   26. Levin, A. M. et al. Exploiting a natural conformational switch    to engineer an interleukin-2 ‘superkine’. Nature 484, 529-533    (2012).-   27. Mitra, S. et al. Interleukin-2 activity can be fine tuned with    engineered receptor signaling clamps. Immunity 42, 826-838 (2015).-   28. Boyman, O. et al. Selectively expanding subsets of T cells in    mice by injection of interleukin-2/antibody complexes: implications    for transplantation tolerance. Transplantation proceedings 44,    1032-1034 (2012).-   29. Tomala, J. et al. Chimera of IL-2 linked to light chain of    anti-IL-2 mAb mimics IL-2/anti-IL-2 mAb complexes both structurally    and functionally. ACS chemical biology 8, 871-876 (2013).-   30. Gutbrodt, K. L., Casi, G. & Neri, D. Antibody-based delivery of    IL2 and cytotoxics eradicates tumors in immunocompetent mice.    Molecular cancer therapeutics 13, 1772-1776 (2014).-   32. Yamane, B. H., Hank, J. A., Albertini, M. R. & Sondel, P. M. The    development of antibody-IL-2 based immunotherapy with hu14.18-IL2    (EMD-273063) in melanoma and neuroblastoma. Expert opinion on    investigational drugs 18, 991-1000 (2009).-   33. Carmenate, T. et al. Human IL-2 mutein with higher antitumor    efficacy than wild type IL-2. J Immunol 190, 6230-6238 (2013).-   34. Heaton, K. M. et al. Characterization of lymphokine-activated    killing by human peripheral blood mononuclear cells stimulated with    interleukin 2 (IL-2) analogs specific for the intermediate affinity    IL-2 receptor. Cellular immunology 147, 167-179 (1993).-   35. Imai, K., Matsuyama, S., Miyake, S., Suga, K. & Nakachi, K.    Natural cytotoxic activity of peripheral-blood lymphocytes and    cancer incidence: an 11-year follow-up study of a general    population. Lancet 356, 1795-1799 (2000).-   36. Lazear, E. et al. Cowpox virus OMCP antagonizes NKG2D via an    unexpected binding orientation. PLos Pathogen In revision (2014).-   37. Deng, W. et al. Antitumor immunity. A shed NKG2D ligand that    promotes natural killer cell activation and tumor rejection. Science    348, 136-139 (2015).-   38. Gorelik, E. & Herberman, R. B. Susceptibility of various strains    of mice to urethan-induced lung tumors and depressed natural killer    cell activity. J Natl Cancer Inst 67, 1317-1322 (1981).-   39. Mancia, F. et al. Optimization of protein production in    mammalian cells with a coexpressed fluorescent marker. Structure 12,    1355-1360 (2004).-   40. Hank, J. A. et al. Distinct clinical and laboratory activity of    two recombinant interleukin-2 preparations. Clin Cancer Res 5,    281-289 (1999).

Introduction to Examples 7-10

Intracellular surveillance mediated by MHC class I (MHCI) is a criticalhost immune function and as such MHCI molecules are frequently targetedfor destruction or intracellular retention by viruses [1]. Manyherpesviruses encode at least one protein that prevents the cell surfaceexpression of MHCI [1,2]. However, this immune evasion strategy rendersthe infected cell susceptible to NK cell-mediated lysis due to loss ofinhibitory signals [3]. Viral infection also leads to cell surfacedisplay of NKG2D ligands (NKG2DLs) recognized by the activating receptorNKG2D, further predisposing the infected cell towards NK cell-mediatedlysis. Therefore, viruses that target MHCI expression often alsosabotage NKG2D-mediated cell responses by targeting NKG2DLs on theinfected cell [4-7].

NKG2DLs are not normally expressed on the cell surface but can beinduced by cellular stress [8]. The specific trigger for NKG2DLexpression is not known but NKG2DLs are upregulated in response toseveral viral infections [9-12]. NKG2DLs comprise a large group ofproteins all recognized by NKG2D, despite having low sequence identity.NKG2DLs include the MIC (A and B) and ULBP (1-6) families in humans aswell as MULT1 and the RAE-1 (α-ε) and H60 (a-c) families in mice [13].The redundancy in NKG2DLs is likely due to a combination of tissuespecific expression patterns of the ligands and the need to counterviral NKG2D evasion strategies [14]. Many viruses have evolvedmechanisms to inhibit the cell surface expression of NKG2DLs as a meansof interfering with NKG2D surveillance of viral infection. This strategyis most apparent among β- and γ-herpesviruses, in which four murinecytomegalovirus proteins (m138, m145, m152, m155) [15-18], two humancytomegalovirus proteins (UL16, UL142) [19,20] and one Kaposi'ssarcoma-associated herpesvirus protein (K5) [21] have been demonstratedto block NKG2DL surface expression. This evasion strategy is also foundin RNA viruses, as hepatitis C virus NS3/4a and human immunodeficiencyvirus Nef proteins also block the expression of a subset of NKG2DLs[22,23]. Additionally, human cytomegalovirus, herpes simplex virus type1 and Epstein-Barr virus each also encode at least one miRNA thatprevents translation of MICB [24,25]. Similarly, JCV and BKV polyomaviruses target ULBP3 with miRNAs [26]. However, blocking NKG2DLexpression on the infected cell is an imperfect evasion strategy, sinceno single viral protein or miRNA has been shown to block the expressionof all NKG2DLs.

Like several herpesviruses, cowpoxvirus (CPXV) also sabotages MHCIexpression. CPXV expresses CPXV012 and CPXV203, two proteins thatprevent TAP-mediated peptide transport and MHCI trafficking to the cellsurface, respectively [27-34]. Ectromelia virus, a relatedorthopoxvirus, induces NKG2DL expression and NKG2D is critical for thecontrol of ectomelia virus pathogensis [35]. Infection with anotherorthopoxvirus, monkeypox virus, leads to dramatic expansion of NK cellsbut impaired NK cell function [36]. Together this suggests that CPXVinfected cells would be sensitive to NK cell-mediated lysis.

Unlike herpesviruses, CPXV does not target NKG2DLs. Instead this virustargets NKG2D directly by encoding a competitive inhibitor of NKG2DLs,orthopoxvirus MHC class I-like protein (OMCP) [37,38]. OMCP is a 152residue protein that is secreted from infected cells and antagonizes theNKG2D-mediated killing of NKG2DL-expressing target cells [37]. OMCP alsoplays an important role in vivo, with OMCP-null CPXV attenuated in mousemodels of infection (M. Sun et al, personal communication). OMCP bindsto murine NKG2D with an affinity equal or greater than all tested murineNKG2DLs, and to human NKG2D with an affinity ˜5,000-fold higher thanhuman NKG2DLs [37-40].

Despite their divergence in sequence identity, all known host NKG2DLsshare common structural features [41,42]. NKG2DLs contain an MHCI-likeplatform domain composed of an eight-stranded beta sheet with twohelices [43-47]. The platform domain is subdivided into α1 and α2domains, with each domain containing four beta strands and an alphahelix. Unlike MHCI, the groove between the helices of the NKG2DLplatform domain is closed and therefore NKG2DLs do not bind peptides.

Like host NKG2DLs, OMCP also adopts an MHCI-like platform domain [38].However, the platform domain of OMCP has been trimmed to have only asix-stranded beta sheet with shorter flanking helices. We termed thehelix of the α1 domain H1 and the discontinuous helix of the α2 domainis termed H2a and H2b. The H2a and H2b helices of OMCP are alsorearranged to be flatter against the beta sheet and to be splayed apartfrom each other. These differences in the OMCP structure werehypothesized to be important for the high affinity binding of OMCP toNKG2D. However, OMCP was still expected to bind to NKG2D in the sameorientation as host NKG2DLs, i.e. with the alpha helices orienteddiagonally within the symmetric NKG2D binding groove.

Here we report the 2.0 Å-resolution structure of human NKG2D bound toOMCP of the Brighton Red strain of cowpoxvirus. The structure reveals asignificant reorientation of OMCP in the NKG2D binding groove relativeto host NKG2DLs. The interface of OMCP with NKG2D is highlycomplementary, buries a significantly larger surface area than hostNKG2DLs, and remains continuous across the entire NKG2D binding groove.This novel binding adaptation and high affinity allows OMCP to competewith the high local concentration of membrane-associated host NKG2DLs.We further show that the mechanism of NKG2D antagonism requires OMCP tobe secreted, lest it lead to NKG2D signaling.

Example 7. Structure Determination of OMCP-NKG2D

We had previously solved the structure of OMCP alone and shown that,similar to host NKG2DLs, OMCP adopts an MHCI-like platform domain [38].Despite the overall similarity of the domain structure of OMCP to hostNKG2DLs, OMCP had several notable deviations in the putativeNKG2D-binding site that were hypothesized to be important for the highaffinity binding of OMCP to NKG2D. To further understand the unusuallyhigh affinity of OMCP for NKG2D, we crystallized and solved thestructure of OMCP bound to human NKG2D.

Initial crystallization trials with OMCP and NKG2D yielded ˜30 differentcrystallization conditions. Subsequent data collection and molecularreplacement of multiple low-resolution crystal forms all yielded similarpartial solutions, with alternating sheets of OMCP-NKG2D complexesseparated by undefined density. In the original structure of OMCP alone,the beta sheets packed to form a trimer with the alpha helices orientedaway from the center [38]. An identical OMCP trimer formed in theOMCP-NKG2D partial solutions, with NKG2D now bound to the outward facinghelices (data not shown). In an attempt to change the lattice packing,we introduced mutations into the beta sheet of OMCP that were designedto break the trimeric interface. These mutations were on the oppositeface of OMCP from the NKG2D binding site to avoid disrupting OMCP-NKG2Dbinding. A mutant form of OMCP (Y23D, F95D) crystallized with NKG2D in anew space group and the crystals diffracted to 2.0 Å (Table 1)(FIG.24A).

The electron density map was continuous and unambiguous throughout allchains of the structure, with the exception of Q108 in OMCP. Thisresidue was situated in the center of the largest loop of OMCP andunambiguous density for this residue was also absent from the structureof OMCP alone [38]. The structure of OMCP bound to NKG2D showed no majordifferences from our previous structure of OMCP alone, with an RMSD forall atoms of 0.8 Å. Likewise, NKG2D was also similar to previous NKG2Dstructures with RMSDs ranging from 0.5-0.9 Å. The 33-34 loop of NKG2D isthe only region of either OMCP or NKG2D that displayed above-average Bfactors. This loop is thought to be flexible and has had above average Bfactors in all previous NKG2D structures [48]. Interestingly, thepeptide bond between S193-S194 in our NKG2D structure had a cisconformation not described in other NKG2D structures (FIG. 29).

Example 8. The Interface Between OMCP and NKG2D

OMCP was hypothesized to bind to the same surface of NKG2D used by hostNKG2DLs because (i) OMCP competed with host NKG2DLs for NKG2D and (ii)mutations within the NKG2DL-binding pocket of NKG2D altered OMCP bindingaffinity [38]. OMCP does bind NKG2D using the same concave bindingpocket as host NKG2DLs (FIG. 24A). OMCP binds primarily using thediscontinuous helices of its α2 domain, H2a and H2b. The position of theH2a and H2b helices is such that every surface exposed side chain ofboth helices within the binding site directly contacts NKG2D (FIG. 24B).Only two contacts are found outside of H2a and H2b, Ile49 and Arg66.Both of these residues are within the α1 domain but lie outside of theH1 helix.

Twelve OMCP residues contact eighteen NKG2D residues to form a mixtureof bond types (Table 2). Three residues in each NKG2D half-site areknown as core binding residues because they make contacts with all knownhost NKG2DLs. The core residues of NKG2D subunit A (NKG2DA) (Tyr152,Tyr199, Met184) form two hydrogen bonds and make extensive hydrophobiccontacts with OMCP residues. The core residues of NKG2D^(A) contact fourOMCP residues and the most critical of these residues is Phe122. Phe122makes multiple hydrophobic contacts with all three NKG2D^(A) coreresidues, including pi-stacking with Tyr152. Phe122 also forms abackbone-to-sidechain hydrogen bond with Tyr152. Interestingly, OMCP isthe first NKG2D ligand not to utilize all six NKG2D core-bindingresidues, with only Met184 and Tyr152 of NKG2D subunit B (NKG2D^(B))contacting OMCP. NKG2DB Met184 and Tyr152 each make a single hydrogenbond and hydrophobic contacts with OMCP residues. Two OMCP residues,Trp127 and Asp132, make contacts with both NKG2D protomers. OMCP Trp127forms a hydrogen bond to Lys150 of NKG2DA and makes several hydrophobiccontacts with Leu148 of NKG2D^(B), Lys150 and Ser151 of NKG2D^(A). OMCPAsp132 forms a hydrogen bond with Tyr152 of NKG2DB and a salt bridgewith Lys150 of NKG2D^(A) (FIG. 25A).

Due to the high affinity of the OMCP-NKG2D interaction we harnessed ahigh throughput in vitro selection approach to find NKG2D-binding nullmutants (Table 3). The results of the screen identified D132 as animportant residue for disrupting NKG2D binding. We then generated themutation D132R in attempt to completely ablate NKG2D binding.Surprisingly, the D132R mutant alone was unable to bind to NKG2D atconcentrations 35-fold above the K_(D) (FIG. 25B), but did not affectbinding of OMCP to FcRL5-expressing cells (FIG. 25C). This mutation islikely to cause significant steric clashes, as well as disrupting bothinteractions made by Asp132 to NKG2D^(A) Lys150 and NKG2D^(B) Tyr152(FIG. 25A).

Previously, the 14-fold higher affinity of OMCP for human vs murineNKG2D was mapped to three amino acid substitutions in the β5′-β5 loop ofNKG2D, abbreviated L2 [38]. In addition to the substitutions themselves(I182V, M184I and Q185P), the position of the loop between NKG2Dorthologs differs. L2 in human NKG2D is bent towards the center of theconcave binding cavity compared to L2 of murine NKG2D. Superimpositionof murine NKG2D onto the human NKG2D-OMCP structure reveals that thecontacts between OMCP and Met184 (mNKG2D residue 1200) in NKG2D^(B) andbetween Met184 (1200) and Glu185 (P201) in NKG2D^(A) would be altereddue to the different position of the murine β5′-β5 loop (FIG. 26A-B).This alteration would disrupt contacts with three residues in OMCP H2a,three residues in H2b and Arg66 within the α1 domain. Of the contactresidues of L2, Met184 makes the most significant contacts in bothNKG2Ds (Table 2)(FIG. 26C). Critically, of the 58 NKG2D sequencesavailable in GenBank, 54 conserve the Met184 and Glu185 found in thehigh affinity human NKG2D (FIG. 26D).

Eighteen OMCP variants have been described between different CPXV andMPXV strains [51]. In this study we have crystallized OMCP from theBrighton Red strain of CPXV which has >60% sequence identity with thehighly conserved sequence of the other 17 OMCP variants, collectivelytermed OMCP_(mpx). Of the 12 OMCP contact residues observed, 9 areidentical to OMCP_(mpx). Of the remaining contacts, all three areconservative hydrophobic substitutions (I49L, T118I and M135I) (FIG.27). OMCPmx binds to NKG2D and the substitutions in the NKG2D contactresidues are unlikely to grossly affect the affinity of OMCPmx for NKG2D[37].

Example 9. A Novel NKG2D-Binding Adaptation

Host NKG2DLs have low sequence identity but overall similar structures,with MHCI-like platform domains binding diagonally across the symmetricbinding groove created by the NKG2D homodimer [13,41,52]. Host ligandscontact one NKG2D half site with H1 and the S1-S2 loop, and contact thesecond NKG2D half site with H2b. Despite the similar MHCI-like fold,OMCP binds the NKG2D binding groove in a novel orientation, rotating 45°relative to host NKG2DLs (FIG. 27). Instead of using H1 and S1-S2 looplike host ligands, OMCP has replaced these contacts with H2a. Thisrotation leads to the helices of OMCP being perpendicular to the NKG2Dbinding groove, instead of lying diagonally across it.

Two unique rearrangements of H2a and H2b make the OMCP orientationpossible. The α2 helices of OMCP and host NKG2DLs are discontinuous,with the two shorter helices hinged relative to each other. For hostligands, the angle between H2a and H2b is ˜90°, positioning H2a awayfrom the NKG2D interface. In contrast, OMCP has increased the hingeangle between the helices by ˜20°, leading to a α2 helix that is flatterrelative to the beta sheet of OMCP. The flattening of the α2 helixallows H2a and H2b to closely complement the concave binding groove ofthe NKG2D homodimer (FIG. 24B). The tight fit of the α2 helix for NKG2Dis reflected in the high shape complementarity (0.77) and buried surfacearea (2,612 Å²). In contrast, host NKG2DLs have shape complementarityranging from 0.63-0.72 and buried surface areas ranging from 1,700-2,180Å² [43,44,46].

The second unique feature of the α2 helix is the separation of H2a andH2b relative to each other. This region also contains a translation thatcompletely separates H2a and H2b into two distinct helices. Thistranslation is critical for NKG2D binding because it allows each helixto be directly centered on the core binding sites of each NKG2D monomer(FIG. 27). This creates a symmetric binding site on OMCP that recognizesthe symmetric binding groove created by the NKG2D dimer. The symmetrybetween OMCP and NKG2D binding is in stark contrast to the canonicalbinding of an asymmetric host ligand to the symmetric NKG2D bindinggroove [52]. However, one element of asymmetry remains in the OMCP-NKG2Dinteraction because each NKG2D half-site recognizes an OMCP helix in adifferent N- to C-terminal orientation, demonstrating again theflexibility of NKG2Ds rigid adaptation recognition [41,53].

The contact sites between NKG2D and host NKG2DLs are made up of twopatches centered on the core binding sites of NKG2D and H1/S1-S2 loopand H2b of NKG2DLs [41]. As a result, the interface of NKG2D withNKG2DLs is discontinuous, particularly in the center of the NKG2Dbinding groove (FIG. 27). Due to the unique orientation of OMCP, H2a andH2b make continuous contacts along the entire NKG2D binding groove (FIG.27). The sidechains of OMCP Lys126, Trp127, Glu131 and Asp132 makecontacts with residues in the center of the NKG2D binding groove andbridge the core binding sites on each NKG2D monomer (FIG. 24B). Inparticular, OMCP Trp127 is directed towards the center of the NKG2Ddimer and makes hydrophobic contacts with residues on both NKG2Dmonomers, effectively closing any gaps in the binding interface.

Example 10. Signaling of NKG2D Upon Ligand Engagement

CPXV and MPXV-infected cells secrete OMCP, which can act as anNKG2D-antagonist [37]. This immune evasion strategy is reminiscent ofcancer induced-NKG2DL shedding. Some cancer cells proteolytically cleaveNKG2DLs from the cell surface using matrix metalloproteinases (MMPs),simultaneously preventing NKG2D-bearing lymphocytes from targeting thecancer cell, as well as creating soluble NKG2DLs to inhibit NKG2D intrans. Cell-associated NKG2DLs trigger NKG2D effector functions (FIG.28A), while cancer-induced, soluble NKG2DLs block NKG2D function (FIG.28B). Like shed NKG2DLs, OMCP is soluble and blocks NKG2D function intrans [37] (FIG. 28C). Unlike host NKG2DLs, OMCP binds NKG2D with anovel orientation.

We therefore asked whether OMCP could serve as a NKG2D agonist in thecontext of the cell membrane, analogously to host NKG2D ligands. SinceOMCP is a secreted protein, an artificially cell-associated OMCP wasconstructed by using a heterologous transmembrane domain from Thy1.1[37] (FIG. 28D). To measure NKG2D-mediated cell killing, we stablytransduced Ba/F3 cells with retroviral vectors expressing either theOMCP-Thy1.1 construct or host NKG2DLs. OMCP-Thy1.1-expressing targetcells were killed equivalently to host NKG2DL-transduced target cells,indicating that despite its altered binding orientation, cell-associatedOMCP was able to activate NKG2D signaling (FIG. 28E). Thus, OMCP must besecreted lest it active NKG2D-effector functions itself, despitepotential loss of efficacy due to diffusion.

Discussion for Examples 7-10

While many viruses have adopted a general mechanism of NKG2D-sabotage bytrying to retain multiple host-encoded NKG2D ligands within the infectedcell, CPXV and MPXV take the very different approach of targeting NKG2Ddirectly. Since NKG2D is monomorphic, this mechanism has the significantadvantage of requiring a single protein to prevent NKG2D recognition ofthe infected cell. The large number of sequence-divergent host NKG2DLsand their associated polymorphisms are thought to be driven by selectionfrom pathogen-encoded NKG2DL antagonists [14]. Likewise, viral NKG2Lantagonists are under selective pressure from the diverse host NKG2DLsin a continual cycle of adaptation. Due to the need to recognizemultiple NKG2DLs, NKG2D has a limited mutational space to adapt. Thelimited ability of NKG2D to mutate is yet another advantage of OMCPdirectly targeting NKG2D, instead of NKG2DLs.

Similarly to OMCP, some cancer cells shed host NKG2DLs to create theirown soluble NKG2D antagonists. However, this strategy has the additionalbenefit of removing host NKG2DL from the surface of cancer cells. Incontrast, CPXV and MPXV lack a known mechanism of blocking host NKG2DLsurface expression. Secreted OMCP must then be able to competeefficiently against the high local concentration of multiple hostNKG2DLs on the infected cell, as well as against diffusion away from theinfected cell. One possible way to increase OMCP's ability to competewith host ligands would be to increase the avidity of OMCP by havingmultiple NKG2D-binding domains. However, a multimeric OMCP couldcrosslink NKG2D and potentially trigger NKG2D-mediated killing.Therefore, secreted OMCP must be monomeric to prevent aberrant NKG2Dsignaling. Thus to compensate for these deficiencies, OMCP must have thehighest affinity possible to effectively compete against cell-associatedhost NKG2DLs [37,38]. The half-life of ligand-receptor interactionscorrelate well with physiological competitiveness [55]. OMCP binds humanand murine NKG2D with half-lives of 348 and 54 seconds, respectively,compared to half-lives of 1.5-18 seconds for most NKG2DLs [38,44,56].Indeed, the increased half-life for NKG2D allows OMCP to effectivelyantagonize NKG2D-mediated immunity in a murine infection model (M. Sunet al, personal communication).

To understand the molecular basis for the long half-life of OMCP forNKG2D, we previously determined the structure of OMCP alone, and here,we report the structure of OMCP bound to NKG2D. The structure of OMCPalone was grossly similar to that of host NKG2D ligands, containing anatypical MHCI-like platform domain. Host NKG2D ligands bind with thehelices of their platform domains oriented diagonally within thesymmetric binding groove of NKG2D. Thus it was expected that OMCP was aviral mimic of host NKG2D ligands and would interact with NKG2Danalogously.

The structure of OMCP-NKG2D instead revealed a novel orientation for anNKG2D ligand in the NKG2D binding groove. Alterations within the α2domain helix allow OMCP to arrange its helices perpendicularly withinthe binding groove. This reorientation places the H2a and H2b helicesdirectly in contact with the core binding sites of NKG2D and also formsthe largest and most continuous binding interface with NKG2D. Becausethe forces (hydrogen bonds, van der Waals, hydrophobic interactions)that mediate protein-protein interactions are individually weak, alarge, continuous interface with high shape complementary allows for acumulatively strong interaction between proteins. This change in thebinding orientation of OMCP reveals how the MHCI-like platform used byhost ligands can be adapted by a pathogen to enhance NKG2D binding.

Since host NKG2DLs and OMCP have a similar MHCI-like platform, it isreasonable to wonder why no host ligand has evolved an analogoushigh-affinity interaction with NKG2D. One likely reason is that the hostimmune response must be carefully calibrated to balance the need forprotection against the threat of autoimmunity. Since the expression ofNKG2DLs on the cell surface signals for effector functions, even a smallamount of high affinity host ligand on the cell surface could trigger animmune response, and the resulting tissue damage could be deleteriousfor the host. Indeed, NKG2D-expressing cells and/or aberrant expressionof host NKG2DLs have been implicated in diabetes, celiac disease andrheumatoid arthritis [57-60]. Viruses are not constrained by autoimmuneselective pressures. Therefore, CPXV and MPXV were free to evolve aviral NKG2DL with the highest possible affinity to maximize immuneevasion potential.

Interestingly, OMCP triggers NKG2D signaling when attached to a targetcell membrane, despite the novel orientation of OMCP relative to hostNKG2DLs. The interaction of host NKG2DLs with the dimeric NKG2D bearsbroad structural similarity to the interaction between MHC moleculeswith their cognate T cell receptors (TCRs). In both cases, theNKG2DL/MHC lies diagonally across the surface created by the dimericNKG2D/TCR. However, there are several examples of MHC-TCR complexesthat, like OMCP-NKG2D, interact with unconventional orientations[61-65]. Several of these complexes involved autoimmune MHC-TCRcomplexes that were tilted or rotated outside of the normal range forMHC-TCR complexes [61,65]. While these receptors could induce TCRsignaling at high MHC concentrations, they failed to assemblecharacteristic immunological synapses [66]. A striking example ofunconventional binding was found when an in vitro peptidelibrary-MHC-TCR (H2-L^(d)-42F3) screen produced a p3A1-H2-L^(d)-42F3complex with an interface rotated 40° relative to other H2-L^(d)-42F3complexes. This rotation places the TCR nearly parallel with the MHCpeptide-binding groove and shifted the interface center almost entirelyon one of the MHC α helices-an orientation strikingly similar to theinterface of OMCP-NKG2D [65]. Interestingly, the p3A1-H2-L^(d)-42F3complex failed to induce TCR signaling [65]. Thus, unlike OMCP/NKG2D,the orientation of MHC relative to TCR is an important factor forsignaling.

OMCP-NKG2D and p3A1-H2-L^(d)-42F3 have opposite signaling outcomes,despite having very similar orientations. TCR signaling requiresco-receptor binding to either the α2/β2 or α3 domains of MHCII or MHCI,respectively. The failure of p3A1-H2-L^(d)-42F3 to signal, and of otherunconventional MHC-TCR complexes to form true immunological synapses, ispotentially due to the inability of co-receptors to form correctquaternary structures for signaling [64,65,67]. Signaling by NKG2D isnot known to require co-receptor stimulation and the majority of NKG2DLslack the co-receptor binding α2/β2 or α3 domains of true MHC molecules.This difference in co-receptor dependency likely explains why OMCP (whenattached via transmembrane) is still competent to stimulateNKG2D-signaling compared to MHC-TCR complexes with unconventionalbinding orientations. Further, it suggests that clustering of NKG2D onthe cell surface is the major determinant of NKG2D-mediated activation.

Methods for Examples 7-10

Identification of NKG2D-binding null mutant D132R. A high throughput invitro selection approach based on combinatorial cell surface display wasutilized to identify NKG2D-binding null mutants. The sequence of OMCPwas globally mutagenized using error-prone PCR, and the mutatedamplicons were spliced to a signal-less Thy1.1 cDNA via overlapextension PCR. This library of mutated OMCPs fused to unmutated Thy1.1was cloned into the pMXs-IRES-EGFP retroviral transfer vector (kind giftof Toshio Kitamura, University of Tokyo) to generate a molecular libraryfor transduction into Ba/F3 cells. The transductants were then sortedfor green fluorescence and anti-Thy1.1 expression to yield a cellularlibrary whose members all had surface expression of OMCP, filtering outmutations giving frameshifts, premature stop codons, andfolding-incompetent OMCP. This OMCP library was sorted for NKG2D bindingusing NKG2D-tetramers. Sorted cells were cloned by limiting dilution andanalyzed. The retroviral cassettes of cells lacking or having reducedNKG2D-binding activity were amplified and sequenced. Utilizing thisapproach, we identified Asp132 as a critical residue for NKG2D binding.

Protein expression and purification. OMCP_(BR) and human NKG2Dexpression constructs were previously described [38]. The (D132R)OMCP_(BR) protein was prepared identically to WT OMCP_(BR). (23D/95D)OMCP-NKG2D complex was reconstituted by oxidative co-refolding frompurified inclusion bodies, as described previously [38]. Refoldedprotein was slowly diluted 10-fold with water and captured on a 5 mlHiTrap Q HP column (GE Healthcare) using a Profinia instrument(Bio-Rad). The captured protein was washed with 50 mM Tris, pH 8.5, 20mM NaCl and bulk eluted with 50 mM Tris, pH 8.5, 250 mM NaCl. The elutedprotein was then concentrated and further purified by gel filtrationchromatography on a Superdex S75 column (16/60; Amersham Biosciences).Fractions containing mono-dispersed OMCP-NKG2D complex (˜50 KDa) werepooled and buffer exchanged into 25 mM Ammonium acetate pH 7.4.

Crystallization, data collection and processing. Native protein crystalswere grown by hanging drop vapor diffusion at 20° C. by streak seedinginto a well solution containing 15% PEG 3350, 0.2M MgCl₂, 0.1M Bis-TrispH 6.75. Crystals were cryoprotected with well solution containing 15%glycerol before flash freezing directly in a liquid nitrogen bath.Diffraction data were collected at the Advanced Light Source synchrotron(beamline 4.2.2). Native (23D/95D) OMCP-hNKG2D crystal diffraction datawere collected at 100 K and at a wavelength of 1.00004 Å. Additionaldiffraction data statistics are summarized in Table 1. Data processingwith HKL2000 [68] showed the crystals belonged to the primitivemonoclinic space group P2₁ (space group #4). The asymmetric unit of thecrystal contained two copies of the (23D/95D) OMCP-hNKG2D complex.

Model building and refinement. The structures of human NKG2D (1MPU) [48]and OMCP (4FFE) [38] were used as search models for molecularreplacement through Phenix [69]. Reiterative refinement and manualrebuilding were performed using Phenix and Coot [70], respectively. Both2Fo-Fc and Fo-Fc maps were used for manual building and to place solventmolecules. The final model yielded an R_(work) of 16.6% and R_(free) of21.4%, with 4% of all reflections set aside for free R factorcross-validation. Progress in refinement was also measured using theMOLPROBITY webserver [71]. The final Ramachandran statistics for themodel were 98% favored and 0% outliers. Additional refinement statisticsare summarized in Table 1. Images of structures were produced using theprogram PyMol [72].

Structure analysis. Analysis of the contact residues, buried surfacearea and shape complementarity of the OMCP-NKG2D interface were carriedout using the programs Ligplot+ [73], PISA [74] and SC [75]. Structuralprograms as compiled by the SBGrid consortium [76]. Analysis of NKG2Dconservation was performed using the ConSurf server [77-80]. GenBanknumbers for species used in Consurf analysis are: Humans (30749494),Borean orangutan (21902299), Chimpanzee (57113989), Gibbon (332232684),Macaque (355785888), Green Monkey (63/506,3485), Common marmoset(380848799), Mouse (148667521), Brown rat (149049263), Guinea Pig(348569092), Ground squirrel (532114387), Deer mouse (589967905), Nakedmole rat (512868733), Prairie vole (532053033), European Shrew(505834608), Star-nosed mole (507978716), Chinese hamster (537136230),and Cat (410963826).

Atomic coordinates. The atomic coordinates (accession code 4PDC) havebeen deposited in the Protein Data Bank, Research Collaboratory forStructural Bioinformatics (Rutgers University, New Brunswick, N.J.)

In vitro NK cell killing assays. Splenocytes from C57B16 mice werepreactivated with 200 U/ml IL-2 for 24 hours and used as cytotoxiceffectors against stably transduced Ba/F3 cell lines in standard killingassays. Target cells were carboxyfluorescein succinimidyl ester (CFSE)labeled and co-incubated with activated splenocytes at 37° C., 5% CO2for 4 hours at effector:target ratios of 10:1, 20:1, and 40:1. Killingpercentage was determined by incorporation of the dead cell exclusiondye 7-amino-actinomycin D (7AAD) in the CFSE+ target population asassessed by flow cytometry. Percent specific lysis was calculated usingthe formula [(experimental dead %−background dead %)/(maximum releasedead %-background dead %)]×100. C57BL/6 mice were obtained from theNational Cancer Institute (Charles River, Mass.). Mice were maintainedunder specific pathogen-free conditions and used between 8 and 12 weeksof age. Single cell suspensions of splenocytes used in killing assayswere generated using standard protocols [81].

TABLE 1 Data collection and refinement statistics OMCP_(BR)-hNKG2D Datacollection Space group P2₁ Cell dimensions a, b, c (Å) 43.3, 101.1, 91.4α, β, γ (°) 90.0, 91.6, 90.0 Resolution (Å) 50-2.0 (2.07-2.00) R_(sym)11.8 (48.5) / / σ 14.5 (3.8) Completeness (%) 93.5 (91.5) Redundancy 6.2(5.3) Refinement Resolution (Å) 44-2.0 Total reflections 309693 Uniquereflection 50139 R_(work) 16.6% (21.0%) R_(free) 21.4% (29.5%) WilsonB-factor 21.62 Protein residues 791 Water molecules 524 R.M.S.deviations Bond lengths (Å) 0.003 Bond angles (°) 0.79 ^(a)As defined byPHENIX [69]

TABLE 2 Interface contacts between NKG2D and OMCP NKG2D-A OMCP Bond typeLys150 Asp132 Salt bridge Lys150 Trp127 H bond Lys150 Trp127 Φ (3)Ser151 Lys126 H bond Ser151 Trp127 Φ (1) Tyr152 Phe122 H bond Tyr152Phe122 Φ (9) Tyr152 Lys126 Φ (5) Met184 Thr118 H bond Met184 Thr119 Φ(1) Met184 Phe122 Φ (5) Gln185 Arg66 Φ (1) Leu191 Phe122 Φ (1) Tyr199Phe122 Φ (4) Glu201 Arg66 Salt bridge Thr205 Arg66 H bond NKG2D-B OMCPBond type Leu148 Trp127 Φ (1) Ser151 Glu131 H bond Tyr152 Asp132 H bondTyr152 Glu131 Φ (3) Tyr152 Met135 Φ (5) Ile182 Ile49 Φ (2) Glu183 Arg142Salt bridge Met184 Met135 Φ (1) Met184 Arg138 Φ (2) Met184 Arg142 H bondLys186 Arg142 Φ (1) Leu191 Met135 Φ (1) Glu201 Arg138 Salt bridgeHydrogen bonds (H bonds), salt bridges and carbon-to-carbon hydrophobicinteractions (Φ) are shown for each contact residue. The number ofhydrophobic interactions between contact residues is designated inparenthesis.

TABLE 3 NKG2D binding mutations identified through global Frequency ofSolvent Amino Acid Mutation Associated Mutations Accessible D132 4 D132N++ D132N, T31S, V68A D132G, K126N, D76V D132G, K126N, D76V K126 4 K126N++ K126N, S71G K126N, D132G, D76V K126N, D132G, D76V K125 2 K125E, F65C− K125E, F92V S120 2 S120Y − S120Y, E10A, N56K D76 2 D76V, D132G, K126N++ D76V, D132G, K126N W116 2 W116R − W116R, K113Q R123 2R123G, D26G, F50L − R123G, D21V, F128L E75 1 E75D − S71 1 S71G, K126N ++F92 1 F92V, K125E + F65 1 F65C, K125E − K113 1 K113Q, W116R + E10 1E10A, N56K, S120Y ++ N56 1 E10A, N56K, S120Y ++ D21 1 D21V, R123G, F128L++ F128 1 D21V, R123G, F128L − D26 1 D26G, F50L, R123G ++ F50 1D26G, F50L, R123G − T31 1 T31S, V68A, D132N ++ V68 1 T31S, V68A, D132N +I30 1 I30L, L51F, L64P, M135T − L51 1 I30L, L51F, L64P, M135T − L64 1I30L, L51F, L64P, M135T ++ M135 1 I30L, L51F, L64P, M135T ++ R67 1R67S, L117P, T119N, F122L + L117 1 R67S, L117P, T119N, F122L − T119 1R67S, L117P, T119N, F122L ++ F122 1 R67S, L117P, T119N, F122L ++Mutations were sequenced from 17 clones expressing mutagenizedOMCP-Thy1.1. Clones were selected for reduced binding to NKG2Dtetramers. The selected clones showed variable deficits in NKG2Dbinding. Each clone had 1-4 mutations in the amino acid sequence of OMCP(5 clones with 1 mutation; 4 clones with 2 mutations; 6 clones with 3mutations; 2 clones with 4 mutations). Silent mutations are notindicated. Mutations are listed in the order of frequency sequenced fromthe selected clones, and mutations that occurred together withinindividual clones are listed where applicable. Clones underlined have atleast one mutation in a solvent inaccessible residue that may alter theoverall stability of OMCP.

REFERENCES FOR EXAMPLES 7-10

-   1. Hansen T H, Bouvier M (2009) MHC class I antigen presentation:    learning from viral evasion strategies. Nat Rev Immunol 9: 503-513.-   2. Griffin B D, Verweij M C, Wertz E J (2010) Herpesviruses and    immunity: the art of evasion. Vet Microbiol 143: 89-100.-   3. Karre K, Ljunggren H G, Piontek G, Kiessling R (1986) Selective    rejection of H-2-deficient lymphoma variants suggests alternative    immune defence strategy. Nature 319: 675-678.-   4. Orange J S, Fassett M S, Koopman L A, Boyson J E, Strominger J    L (2002) Viral evasion of natural killer cells. Nat Immunol 3:    1006-1012.-   5. Lisnic V J, Krmpotic A, Jonjic S (2010) Modulation of natural    killer cell activity by viruses. Curr Opin Microbiol 13: 530-539.-   6. Finton K A, Strong R K (2012) Structural insights into activation    of antiviral N K cell responses. Immunol Rev 250: 239-257.-   7. Li Y, Mariuzza R A (2014) Structural Basis for Recognition of    Cellular and Viral Ligands by N K Cell Receptors. Front Immunol    5:123.-   8. Raulet D H (2003) Roles of the NKG2D immunoreceptor and its    ligands. Nat Rev Immunol 3: 781-790.-   9. Draghi M, Pashine A, Sanjanwala B, Gendzekhadze K, Cantoni C, et    al. (2007) NKp46 and NKG2D recognition of infected dendritic cells    is necessary for N K cell activation in the human response to    influenza infection. J Immunol 178: 2688-2698.-   10. Pappworth I Y, Wang E C, Rowe M (2007) The switch from latent to    productive infection in epstein-barr virus-infected B cells is    associated with sensitization to N K cell killing. J Virol 81:    474-482.-   11. Welte S A, Sinzger C, Lutz S Z, Singh-Jasuja H, Sampaio K L, et    al. (2003) Selective intracellular retention of virally induced    NKG2D ligands by the human cytomegalovirus UL16 glycoprotein. Eur J    Immunol 33: 194-203.-   12. Ward J, Bonaparte M, Sacks J, Guterman J, Fogli M, et al. (2007)    HIV modulates the expression of ligands important in triggering    natural killer cell cytotoxic responses on infected primary T-cell    blasts. Blood 110: 1207-1214.-   13. Obeidy P, Sharland A F (2009) NKG2D and its ligands. Int J    Biochem Cell Biol 41: 2364-2367.-   14. Eagle R A, Trowsdale J (2007) Promiscuity and the single    receptor NKG2D. Nat Rev Immunol 7: 737-744.-   15. Lodoen M, Ogasawara K, Hamerman J A, Arase H, Houchins J P, et    al. (2003) NKG2D-mediated natural killer cell protection against    cytomegalovirus is impaired by viral gp40 modulation of retinoic    acid early inducible 1 gene molecules. J Exp Med 197:1245-1253.-   16. Lodoen M B, Abenes G, Umamoto S, Houchins J P, Liu F, et    al. (2004) The cytomegalovirus m155 gene product subverts natural    killer cell antiviral protection by disruption of H60-NKG2D    interactions. J Exp Med 200: 1075-1081.-   17. Krmpotic A, Hasan M, Loewendorf A, Saulig T, Halenius A, et    al. (2005) N K cell activation through the NKG2D ligand MULT-1 is    selectively prevented by the glycoprotein encoded by mouse    cytomegalovirus gene m145. J Exp Med 201: 211-220.-   18. Lenac T, Budt M, Arapovic J, Hasan M, Zimmermann A, et    al. (2006) The herpesviral Fc receptor fcr-1 down-regulates the    NKG2D ligands MULT-1 and H60. J Exp Med 203: 1843-1850.-   19. Cosman D, Mullberg J, Sutherland C L, Chin W, Armitage R, et    al. (2001) ULBPs, novel MHC class I-related molecules, bind to CMV    glycoprotein UL16 and stimulate N K cytotoxicity through the NKG2D    receptor. Immunity 14: 123-133.-   20. Chalupny N J, Rein-Weston A, Dosch S, Cosman D (2006)    Down-regulation of the NKG2D ligand MICA by the human    cytomegalovirus glycoprotein UL142. Biochem Biophys Res Commun    346:175-181.-   21. Thomas M, Boname J M, Field S, Nejentsev S, Salio M, et    al. (2008) Down-regulation of NKG2D and NKp80 ligands by Kaposi's    sarcoma-associated herpesvirus K5 protects against N K cell    cytotoxicity. Proc Natl Acad Sci USA 105: 1656-1661.-   22. Cerboni C, Neri F, Casartelli N, Zingoni A, Cosman D, et    al. (2007) Human immunodeficiency virus 1 Nef protein downmodulates    the ligands of the activating receptor NKG2D and inhibits natural    killer cell-mediated cytotoxicity. J Gen Virol 88: 242-250.-   23. Wen C, He X, Ma H, Hou N, Wei C, et al. (2008) Hepatitis C virus    infection downregulates the ligands of the activating receptor    NKG2D. Cell Mol Immunol 5: 475-478.-   24. Stem-Ginossar N, Elefant N, Zimmermann A, Wolf D G, Saleh N, et    al. (2007) Host immune system gene targeting by a viral miRNA.    Science 317: 376-381.-   25. Nachmani D, Stem-Ginossar N, Sarid R, Mandelboim O (2009)    Diverse herpesvirus microRNAs target the stress-induced immune    ligand MICB to escape recognition by natural killer cells. Cell Host    Microbe 5: 376-385.-   26. Bauman Y, Nachmani D, Vitenshtein A, Tsukerman P, Drayman N, et    al. (2011) An identical miRNA of the human JC and BK polyoma viruses    targets the stress-induced ligand ULBP3 to escape immune    elimination. Cell Host Microbe 9: 93-102.-   27. Gainey M D, Rivenbark J G, Cho H, Yang L, Yokoyama W M (2012)    Viral MHC class I inhibition evades CD8+ T-cell effector responses    in vivo but not CD8+ T-cell priming. Proc Natl Acad Sci USA 109:    E3260-3267.-   28. Byun M, Verweij M C, Pickup D J, Wiertz E J, Hansen T H, et    al. (2009) Two mechanistically distinct immune evasion proteins of    cowpox virus combine to avoid antiviral CD8 T cells. Cell Host    Microbe 6: 422-432.-   29. Byun M, Wang X, Pak M, Hansen T H, Yokoyama W M (2007) Cowpox    virus exploits the endoplasmic reticulum retention pathway to    inhibit MHC class I transport to the cell surface. Cell Host Microbe    2: 306-315.-   30. McCoy W Ht, Wang X, Yokoyama W M, Hansen T H, Fremont D H (2013)    Cowpox virus employs a two-pronged strategy to outflank MHCI antigen    presentation. Mol Immunol.-   31. McCoy W Ht, Wang X, Yokoyama W M, Hansen T H, Fremont D H (2012)    Structural mechanism of E R retrieval of MHC class I by cowpox. PLoS    Biol 10: el001432.-   32. Alzhanova D, Edwards D M, Hammarlund E, Scholz I G, Horst D, et    al. (2009) Cowpox virus inhibits the transporter associated with    antigen processing to evade T cell recognition. Cell Host Microbe 6:    433-445.-   33. Dasgupta A, Hammarlund E, Slifka M K, Fruh K (2007) Cowpox virus    evades CTL recognition and inhibits the intracellular transport of    MHC class I molecules. J Immunol 178:1654-1661.-   34. Luteijn R D, Hoelen H, Kruse E, van Leeuwen W F, Grootens J, et    al. (2014) Cowpox Virus Protein CPXV012 Eludes CTLs by Blocking ATP    Binding to TAP. J Immunol 193:1578-1589.-   35. Fang M, Lanier L L, Sigal L J (2008) A role for NKG2D in N K    cell-mediated resistance to poxvirus disease. PLoS Pathog 4: e30.-   36. Song H, Josleyn N, Janosko K, Skinner J, Reeves R K, et    al. (2013) Monkeypox virus infection of rhesus macaques induces    massive expansion of natural killer cells but suppresses natural    killer cell functions. PLoS One 8: e77804.-   37. Campbell J A, Trossman D S, Yokoyama W M, Carayannopoulos L    N (2007) Zoonotic orthopoxviruses encode a high-affinity antagonist    of NKG2D. J Exp Med 204:1311-1317.-   38. Lazear E, Peterson L W, Nelson C A, Fremont D H (2013) Crystal    structure of the cowpox virus-encoded NKG2D ligand OMCP. J Virol 87:    840-850.-   39. Carayannopoulos L N, Naidenko O V, Kinder J, Ho E L, Fremont D    H, et al. (2002) Ligands for murine NKG2D display heterogeneous    binding behavior. Eur J Immunol 32: 597-605.-   40. Mistry A R, O'Callaghan C A (2007) Regulation of ligands for the    activating receptor NKG2D. Immunology 121: 439-447.-   41. Strong R K, McFarland B J (2004) NKG2D and Related    Immunoreceptors. Adv Protein Chem 68: 281-312.-   42. Deng L, Mariuzza R A (2006) Structural basis for recognition of    MHC and MHC-like ligands by natural killer cell receptors. Semin    Immunol 18: 159-166.-   43. Li P, McDermott G, Strong R K (2002) Crystal structures of    RAE-1beta and its complex with the activating immunoreceptor NKG2D.    Immunity 16: 77-86.-   44. Li P, Morris D L, Willcox B E, Steinle A, Spies T, et al. (2001)    Complex structure of the activating immunoreceptor NKG2D and its MHC    class I-like ligand MICA. Nat Immunol 2: 443-451.-   45. Li P, Willie S T, Bauer S, Morris D L, Spies T, et al. (1999)    Crystal structure of the MHC class I homolog MIC-A, a gammadelta T    cell ligand. Immunity 10: 577-584.-   46. Radaev S, Rostro B, Brooks A G, Colonna M, Sun P D (2001)    Conformational plasticity revealed by the cocrystal structure of    NKG2D and its class I MHC-like ligand ULBP3. Immunity 15:1039-1049.-   47. Adams E J, Luoma A M (2013) The adaptable major    histocompatibility complex (MHC) fold: structure and function of    nonclassical and MHC class I-like molecules. Annu Rev Immunol 31:    529-561.-   48. McFarland B J, Kortemme T, Yu S F, Baker D, Strong R K (2003)    Symmetry recognizing asymmetry: analysis of the interactions between    the C-type lectin-like immunoreceptor NKG2D and MHC class I-like    ligands. Structure 11: 411-422.-   49. Stewart D E, Sarkar A, Wampler J E (1990) Occurrence and role of    cis peptide bonds in protein structures. J Mol Biol 214: 253-260.-   50. Craveur P, Joseph A P, Poulain P, de Brevem A G, Rebehmed    J (2013) Cis-trans isomerization of omega dihedrals in proteins.    Amino Acids 45: 279-289.-   51. Lefkowitz E J, Upton C, Changayil S S, Buck C, Traktman P, et    al. (2005) Poxvirus Bioinformatics Resource Center a comprehensive    Poxviridae informational and analytical resource. Nucleic Acids Res    33: D311-316.-   52. Strong R K (2002) Asymmetric ligand recognition by the    activating natural killer cell receptor NKG2D, a symmetric    homodimer. Mol Immunol 38:1029-1037.-   53. Radaev S, Sun P D (2003) Structure and function of natural    killer cell surface receptors. Annu Rev Biophys Biomol Struct 32:    93-114.-   54. Campbell J A, Davis R S, Lilly L M, Fremont D H, French A R, et    al. (2010) Cutting edge: FcR-like 5 on innate B cells is targeted by    a poxvirus MHC class I-like immunoevasin. J Immunol 185: 28-32.-   55. Copeland R A, Pompliano D L, Meek T D (2006) Drug-target    residence time and its implications for lead optimization. Nat Rev    Drug Discov 5: 730-739.-   56. O'Callaghan C A, Cerwenka A, Willcox B E, Lanier L L, Bjorkman P    J (2001) Molecular competition for NKG2D: H60 and RAE1 compete    unequally for NKG2D with dominance of H60. Immunity 15: 201-211.-   57. Groh V, Bruhl A, E I-Gabalawy H, Nelson J L, Spies T (2003)    Stimulation of T cell autoreactivity by anomalous expression of    NKG2D and its MIC ligands in rheumatoid arthritis. Proc Natl Acad    Sci USA 100: 9452-9457.-   58. Hue S, Mention J J, Monteiro R C, Zhang S, Cellier C, et    al. (2004) A direct role for NKG2D/MICA interaction in villous    atrophy during celiac disease. Immunity 21: 367-377.-   59. Meresse B, Chen Z, Ciszewski C, Tretiakova M, Bhagat G, et    al. (2004) Coordinated induction by IL15 of a TCR-independent NKG2D    signaling pathway converts CTL into lymphokine-activated killer    cells in celiac disease. Immunity 21: 357-366.-   60. Ogasawara K, Hamerman J A, Hsin H, Chikuma S, Bour-Jordan H, et    al. (2003) Impairment of N K cell function by NKG2D modulation in    NOD mice. Immunity 18: 41-51.-   61. Hahn M, Nicholson M J, Pyrdol J, Wucherpfennig K W (2005)    Unconventional topology of self peptide-major histocompatibility    complex binding by a human autoimmune T cell receptor. Nat Immunol    6: 490-496.-   62. Sethi D K, Schubert D A, Anders A K, Heroux A, Bonsor D A, et    al. (2011) A highly tilted binding mode by a self-reactive T cell    receptor results in altered engagement of peptide and MHC. J Exp Med    208: 91-102.-   63. Wucherpfennig K W, Call M J, Deng L, Mariuzza R (2009)    Structural alterations in peptide-MHC recognition by self-reactive T    cell receptors. Curr Opin Immunol 21: 590-595.-   64. Yin Y, Li Y, Mariuzza R A (2012) Structural basis for    self-recognition by autoimmune T-cell receptors. Immunol Rev 250:    32-48.-   65. Adams J J, Narayanan S, Liu B, Bimbaum M E, Kruse A C, et    al. (2011) T cell receptor signaling is limited by docking geometry    to peptide-major histocompatibility complex. Immunity 35: 681-693.-   66. Schubert D A, Gordo S, Sabatino J J, Jr., Vardhana S, Gagnon E,    et al. (2012) Self-reactive human CD4 T cell clones form unusual    immunological synapses. J Exp Med 209: 335-352.-   67. Li Y, Yin Y, Mariuzza R A (2013) Structural and biophysical    insights into the role of CD4 and CD8 in T cell activation. Front    Immunol 4: 206.-   68. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction    data collected in oscillation mode. Macromolecular Crystallography,    Pt A 276: 307-326.-   69. Adams P D, Grosse-Kunstleve R W, Hung L W, loerger T R, McCoy A    J, et al. (2002) PHENIX: building new software for automated    crystallographic structure determination. Acta Crystallogr D Biol    Crystallogr 58:1948-1954.-   70. Emsley P, Cowtan K (2004) Coot: model-building tools for    molecular graphics. Acta Crystallogr D Biol Crystallogr 60:    2126-2132.-   71. Chen V B, Arendall W B, 3rd, Headd J J, Keedy D A, Immormino R    M, et al. (2010) MolProbity: all-atom structure validation for    macromolecular crystallography. Acta Crystallogr D Biol Crystallogr    66: 12-21.-   72. Schrodinger, LLC (2010) The PyMOL Molecular Graphics System,    Version 1.3r1.-   73. Laskowski R A, Swindells M B (2011) LigPlot+: multiple    ligand-protein interaction diagrams for drug discovery. J Chem Inf    Model 51: 2778-2786.-   74. Krissinel E, Henrick K (2007) Inference of macromolecular    assemblies from crystalline state. J Mol Biol 372: 774-797.-   75. Lawrence M C, Colman P M (1993) Shape complementarity at    protein/protein interfaces. J Mol Biol 234: 946-950.-   76. Morin A, Eisenbraun B, Key J, Sanschagrin P C, Timony M A, et    al. (2013) Collaboration gets the most out of software. Elife 2:    e01456.-   77. Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N (2010) ConSurf    2010: calculating evolutionary conservation in sequence and    structure of proteins and nucleic acids. Nucleic Acids Res 38:    W529-533.-   78. Landau M, Mayrose, Rosenberg Y, Glaser F, Martz E, et al. (2005)    ConSurf 2005: the projection of evolutionary conservation scores of    residues on protein structures. Nucleic Acids Res 33: W299-302.-   79. Glaser F, Pupko T, Paz I, Bell R E, Bechor-Shental D, et    al. (2003) ConSurf: identification of functional regions in proteins    by surface-mapping of phylogenetic information. Bioinformatics 19:    163-164.-   80. Celniker G, Nimrod G, Ashkenazy H, Glaser F, Martz E, et    al. (2013) ConSurf: Using Evolutionary Data to Raise Testable    Hypotheses about Protein Function. Israel Journal of Chemistry    53:199-206.-   81. Dokun A O, Kim S, Smith H R, Kang H S, Chu D T, et al. (2001)    Specific and nonspecific N K cell activation during virus infection.    Nat Immunol 2: 951-956.

Example 11. Individuals with Poorly Functioning Natural Killer Cells areMore Susceptible to Malignancies

FIG. 14A shows that AJ and 129 are lung cancer susceptible strain ofmice and B6 and C3H are lung cancer resistant strains of mice based onthe larger tumor burden found in AJ and 129 mice. FIG. 14B shows thatwhen NK cells from the various mouse strains were incubated with LM2lung carcinoma cells at varying ratios, the NK cells freshly isolatedfrom B6 and C3H mice (lung cancer resistant strains) resulted insignificantly more lysis of LM2 lung carcinoma cells than the NK cellsfreshly isolated from AJ and 129 mice (lung cancer susceptible strains).Taken together these data show that strains of mice that are resistantto lung cancer have NK cells that more effectively lyse lung carcinomacells. Further, susceptible strains have poorly functioning NK cells.

That data also correlates with human data. FIG. 15 shows that a greaterpercentage of NK cells appear to produce TNFα in “resistant” patientsversus “susceptible” patients. Further, it has been shown that tumorsdownregulate the lytic capacity of NK cells, even if they were highlyfunctional before.⁵³ Thus, even individuals with highly functioning NKcells may benefit from therapy to enhance NK cell function.

Notably, ex vivo cytokine activation can reverse natural killer celldysfuction. FIG. 16 shows that IL-2 activated NK cells from bothresistant (B6 and C3H) and susceptible (AJ and 129) mouse strains canlyse LM2 lung cancer cells. Accordingly, mouse NK Cells that did notshow significant lysis of cancer cells (NK cells from 129 & AJ strains)were much more effective at lysis when treated with IL-2. NK cells fromcancer-resistant strains also showed increase % of specific lysis.

Example 12. OMCP-mutIL2 Mediated Immunotherapy In Vivo

Immunoregulation of malignancies involves an intricate interplay ofmultiple cellular components. CD4⁺Foxp3⁺ T_(regs) have been shown inmultiple models to contribute to tumor-specific tolerance and facilitatetumor growth^(4,16,17). NK cells and CD8⁺ CTLs contribute toimmunoregulation of multiple tumors, such as melanoma^(12,18). Othertumors, such as lung cancer, are controlled almost exclusively by NKcells with little contribution by the adaptive immune system^(19,20)(and unpublished data AS. Krupnick). In order to test OMCP-mutIL2mediated immunotherapy we will rely on B16 melanoma expressing the modeltumor antigen ovalbumin (MOS tumor cell line)²¹. Multiple studies havedemonstrated a role for both NK cells and CD8⁺ CTLs in controllingmelanoma growth²²⁻²⁴. Thus the melanoma model offers an experimentaladvantage in studying OMCP-mutIL2, which can activate both types ofcells (FIG. 1E-F). Reagents specific to this tumor, such as tetramersfor the MHC Class I-restricted CD8⁺ T cell receptor specific for themelanoma tumor associated antigen tyrosinase-related protein 2 peptideSVYDFFVWL (SEQ ID NO:3), can be readily purchased commercially(Proimmune, Sarasota, Fl.). The use of an ovalbumin-expressing cell linealso offers the advantage of studying the immune response to a thehighly immunogenic peptide SIINFEKL (SEQ ID NO:4) in addition tonaturally occurring tumor associated antigens such as tyrosinase-relatedprotein 2 which generally expands T cells with low avidity^(25,26).

In order to perform the studies B6 mice will be injected subcutaneouslywith 1×10⁶ MO5 melanoma cells. One week after tumor injection mice willbe divided into 4 groups (10 mice per group) and treated with ten twicea day injections of either: wild type IL2 (group #1); mutIL2 (group #2);OMCP-mutIL2 (group #3) or; saline (group #4) (FIG. 12). Tumor growthwill be followed by daily measurements of diameter for 4 weeks, or untilone of the groups develops tumors >2 cm in diameter. At that point micein all groups will be sacrificed for analysis. In addition to tumorgrowth, lymphocyte infiltration of both the tumor and draining inguinallymph node will be evaluated by flow cytometry. We will quantitate thetotal number and activation status of CD4⁺Foxp3⁺ Tregs (evaluated byICOS and GITR upregulation). We will also evaluate NK cell number andactivation as measured by IFN-γ production and CD69 upregulation.

Antigen-specific CTL generation will be evaluated by quantitating bothCD8⁺ T cells and CD8⁺CD44^(hi)CD62L^(low) effector cells (ECs) that areprimarily responsible for tumor clearance^(22,27,28). Antigenspecificity will be determined by identifying CD8⁺ CTLs with T cellreceptor specific for either the ovalbumin peptide SIINFEKL (SEQ IDNO:4) or melanoma specific tyrosinase-related protein 2 peptideSVYDFFVWL (SEQ ID NO:3) (both tetramers from Proimmune, Sarasota, Fl.).Tumor apoptosis will be quantitated by TUNEL staining.

Based on our in vitro tumor data and in vivo phenotypic analysis wesuspect that the OMCP-mutIL2 group will demonstrate attenuation in tumorgrowth with high number of NK cells, antigen-specific CTLs, specificallyCD8⁺ ECs, and fewer CD4⁺Foxp3⁺ T_(regs). If this turns out to be thecase we would determine the relative role for CD8⁺ or NK CTLs bydepletion experiments. Even if CTLs increase it is possible that MOSgrowth will not be altered. If that turns out to be the case we wouldlook in closer detail at the CD4⁺Foxp3⁺T_(regs) or in the presence andactivation of myeloid-derived suppressor cells in OMCP-mutIL2 treatedmice. Based on melanoma data additional tumors will be tested usingsimilar methods.

Example 13. CD8⁺ Memory T Cell Generation after Treatment withOMCP-mutIL2 Fusion Construct

Once activation through their T cell receptor, naive CD8⁺ T cellsprimarily differentiate into short-lived CD44^(hi)CD62L^(Low) effectorcells (ECs) with cytolytic potential. A portion of activated cells,however, differentiate to long-lived CD44^(hi)CD62L^(hi) central memoryT cells (CD8⁺ CMs)²⁹⁻³¹. CD8⁺CMs act as an antigen specific reservoirfor cellular protection and upon restimulation differentiate intoCD8⁺ECs with cytolytic function. The durability of CD8⁺ CMs makes theman ideal target for ex vivo generation and adoptive transfer forlong-term protection³¹. The possibility of generating this cellpopulation in vivo offers multiple advantages over an ex vivo system,including establishing a polyclonal population reactive to multipletumor associated antigens and avoidance of costs associated with donorpheresis and ex vivo expansion. In vivo expansion of tumor antigenspecific CD8⁺CMs could also eliminate the need for frequent pheresis andcell readministration.

High dose IL2 therapy results in activation of both CD4⁺Foxp3⁺T_(regs)and CD8⁺ T cells but its effect on tumor associated antigen specificCD8⁺CM generation is unknown. Some have demonstrated, using antibodydepletion, that CD4⁺Foxp3⁺T_(regs) interfere with tumor specific CD8⁺CMgeneration^(17,32) while others, using different models, havedemonstrated that CD4⁺Foxp3⁺T_(reg) depletion impairs CD8⁺ memoryformation^(33,34). OMCP-mutIL2 creates a unique immunologic environmentwhere CD4⁺Foxp3⁺T_(regs) are maintained but not actively expanded (FIG.3). While NKG2D is not expressed on resting CD8⁺ T cells, it is inducedon this population upon activation³⁵. Thus, unlike mutIL2, OMCP-mutIL2results in CD8⁺ T cell proliferation at levels comparable to wild-typeIL2 in NKG2D-sufficient mice (FIG. 1E-F). The effect of OMCP-mutII2 onCD8⁺ T cell memory formation, however, is unknown but is critical todecipher based on the long-term tumor specific immunity that this cellpopulation can confer.

In order to test long-term memory formation after cytokine stimulationin vivo we will utilize a model of irradiated tumor cell vaccination andcytokine treatment. In order to accomplish this we will subcutaneouslyinject 1×10⁷ lethally irradiated (10Gy) MOS melanoma cells into C57Bl/6mice. The recipient mice will then be treated either regular IL2 (group#1), mutIL2 (group #2), OMCP-mutIL2 (group #3) or saline (group #4) intwice daily doses over a course of 5 days (FIG. 13). The mice will besacrificed at various time points ranging from one to three months postinfection (FIG. 13). Antigen-specific CD8⁺ CM formation will be assessedby phenotypic analysis of splenic, peripheral lymph node, lung, andliver-resident CD8⁺CD44^(hi)CD62L^(hi) CMs. Antigen specificity will bedetermined by MHC Class I staining for either the ovalbumin peptide,SIINFEKL (SEQ ID NO:4), or melanoma specific tyrosinase-related protein2 peptide SVYDFFVWL (SEQ ID NO:3) (both from Proimmune, Sarasota, Fl.).

In order to test the functional protection of such vaccination protocolsin a separate set of experiments mice from the four groups describedabove will not be sacrificed for phenotypic analysis and will bereinjected with live MOS melanoma (1×10⁶ cells/mouse subcutaneously).Melanoma growth will be assessed by serial measurement of tumordiameter. Contribution of CD8⁺ T cells to any immunologic protectionwill be assessed by CD8-specific antibody depletion in a portion of mice(clone YTS 169.4, BioXcell Inc., West Lebanon, N.H.).

Example 14. Mechanism of CTL Activation by OMCP-mutIL2 Fusion Construct

A mechanistic understanding of the enhanced activation of effector cellfunction by the OMCP-mutIL2 chimera will be critical for optimizing thistherapeutic agent. The interaction of the fusion protein with IL2R andNKG2D are likely to be dependent on several factors including the lengthof the linker peptide (FIG. 1E-F). Therefore, it is critical tounderstand the mechanism of OMCP-mutIL2 chimera mediated CTL activationin order to allow for optimization of the construct and design of futureimmunotherapy protocols. The two-domain chimeric protein couldpotentially increase the activation of NKG2D-expressing cells by threenon-mutually exclusive mechanisms. First and foremost the OMCP-mutIL2construct could increase the avidity of mutIL2 binding to targetedcells. This could lead to an increase in the number of receptorsoccupied and increased signaling intensity compared to mutIL2.Additionally dual binding to both NKG2D and IL2R could decrease the rateof receptor internalization and increase the duration of signaling byIL2. It is also possible that the OMCP-mutIL2 construct alters thesignaling profile by the target cell by activating both the IL2 andNKG2D stimulatory pathways. These three non-mutually exclusive effectscould explain the increase in activation of our construct of CTLs in anNKG2D-mediated fashion.

There are several methods for determining the avidity of a protein for acell, either directly (radiolabeled, fluorescent) or indirectly (antigenexclusion)³⁹. We plan to determine the avidity of wild-type IL2, mutIL2,or OMCP-mutIL2 for CD4⁺Foxp3⁺ T_(regs), NK cells, and CD8⁺ T lymphocytesusing KinExA⁴⁰. To accomplish this we will isolate cells fromsplenocytes of either wild-type C57BI/6 or NKG2D⁴mice on a C57BI/6background using a magnetic bead isolation kit (Miltenyi Biotech, SanDiego, Ca.). Target cells will be serially diluted by a factor of 2 in11 falcon tubes in media containing 0.05% NaN₃. The 12th tube willcontain just the media. OMCP-mutIL2 or mutIL2 alone will then be addedto each tube of either wild-type or NKG2D^(−/−) cells and the cells withcytokine will be rotated at 4° C. for 36 h. At the end of 36 h, thecells were centrifuged at 2400 rpm for 4 min and the free constructpresent in the supernatant will be measured by an anti-IL2 ELISA. Theequilibrium dissociation constant (K_(d)) will then be calculated. Thisapproach has the advantage of measuring the avidity of cell surfacemolecules at physiologic densities and obviates the need for labeling,which can artificially lower the affinity of antibodies for theirantigens^(42,43).

The two-domain structure of the fusion protein is likely tosignificantly increase the half-life of the protein on the surface ofNKG2D⁺ and IL2R⁺ cells. Any increase in surface half-life likely affectsboth the internalization of the bound receptors and signaling intensityand duration. To address the internalization of receptors, we willincubate each construct with the above mentioned cell types over a rangeof times and monitor the change in cell surface expression of IL2Rβγ andNKG2D using flow cytometry as previously described⁴⁴. Of key interestwill be the signaling profile of each construct. IL2-IL2R engagementsignals through JAK-STAT pathways, while NKG2D signals through DAP10/12pathways. While monomeric, soluble OMCP does not induce NKG2D signaling,OMCP can signal when concentrated locally on the cell surface⁴⁵.Therefore, it is critical to determine whether the chimera is capable ofinducing dual signaling through IL2R and NKG2D. IL2-mediated signalingwill be assessed by Western blot for phosphorylated JAK1 and JAK3 infreshly isolated CD4+Foxp3+ T_(regs), NK cells or CD8⁺ T cells incubatedin vitro with the construct^(46,47). NKG2D-mediated signaling will beassessed by immunoprecipitation of DAP10 or DAP12 followed by Westernblotting for phosphotyrosine as previously described^(48,49).

Both IL2 and OMCP interact with their cognate receptors with highaffinity; the fusion of the two proteins is anticipated to greatlyenhance the avidity of the chimeric construct for cells expressing bothIL2R and NKG2D. As a consequence, the tethering of the construct to twocell surface receptors may lead to reduced internalization and increasedduration of signaling. Combined these two phenomena represent the mostlikely mechanism for increased proliferation of NK cells in vivo. Thesignaling via NKG2D relies upon receptor clustering⁴⁵. Since theconstruct is soluble it is possible, though unlikely, that the chimerawill cluster NKG2D and induce DAP10/12 signaling. However, shouldDAP10/12 signaling be detected, we will then investigate the importanceof this signaling in the expansion of NK cells using cells derived fromVav1 knockout mice. Vav1 is a signal mediator downstream of DAP10⁵⁰.Using a Vav1 knockout has the advantage of leaving NKG2D expressionintact, in contrast to DAP knockouts⁵⁰. This will remove the NKG2Dsignaling component while leaving the NKG2D-dependent targeting intact.A clearer understanding of the mechanism of action for OMCP-IL2 chimeradependent expansion will be crucial for further refinements of thetherapeutic agent. Understanding these parameters will allow for testingof different construct designs, primarily in the length of the linkerbetween OMCP and IL2, to calibrate the effects of the chimera.

Example 15. In Vivo Immunotherapy with IL-2, R38 Å/F42K IL2 or OMCPTargeted IL2 Constructs

In order to determine if our construct plays a role in immunoregulationof malignancies as well as viral infections we will rely on in vivomodels of B16 melanoma and mouse cytomegalovirus (MCMV). In one set ofexperiments B6 mice will be injected subcutaneously with 1×10⁶ cells ofthe poorly immunogenic B16 melanoma cell line. One week after tumorinjection mice will be divided into 13 groups (5 mice per group) andtreated with five daily injections of IL2, R38 Å/F42K IL2, OMCP fusionconstructs or saline as described in FIG. 18 and Table 4. Tumor growthwill be followed by daily measurements of diameter for 4 weeks or untilone of the groups develops tumors >2 cm in diameter. At that point micein all groups will be sacrificed for analysis. In addition to tumorgrowth lymphocyte infiltration of both the tumor and draining inguinallymph node will be evaluated by flow cytometry. We will quantitate thetotal number and activation status of CD4⁺Foxp3⁺ Tregs (expressed as %of tumor infiltrating lymphocytes and % ICOS⁺). We will also evaluate NKcell number and activation as measured by IFN-γ production and CD69upregulation. Tumor apoptosis will be evaluated by TUNEL staining.

TABLE 4 Experimental Design for Dosing for IL2, R38A/F42K IL2,OMCP-R38A/F42K IL2 or OMCP linked IL2 constructs LOW INTERMEDIATE HIGHDose Cytokine DOSE DOSE DOSE IL2 Group 1 Group 2 Group 3 R38A/F42K IL2Group 4 Group 5 Group 6 OMCP-wild-type IL2 Group 7 Group 8 Group 9OMCP-R38A/ Group 10 Group 11 Group 12 F42K IL2 Saline Group 13

In order to evaluate the therapeutic potential of IL2 in an infectiousdisease model, B6 mice will be infected with a sublethal dose of MCMV(5×10⁴) particle forming units (PFUs) as previously described²⁹. Day 1post infection the mice will be divided into 13 groups (5 mice pergroup) and treated with five daily injections of IL2, R38 Å/F42K IL2,OMCP fusion constructs or saline as described in FIG. 18 and Table 4. Onpost-infection day #6 the mice will be sacrificed and splenic andpulmonary viral load determined by standard plaque assay.

We expect that treatment with pure IL2 will have little effect on tumorgrowth or viral load as we expect to see preferential activation ofT_(regs) over CTLs. We suspect that administration of the mutant R38Å/F42K form of IL2 will result a lower tumor and viral burden comparedto wild-type IL2 due to less activation of CD4⁺Foxp3⁺ T_(regs).Nevertheless it is possible that despite lower levels of T_(reg)activation the tumor burden will be identical between IL2 and R38 Å/F42KIL2 due to decreased NK activation by the mutant form of IL2 as well. Weexpect OMCP IL2 construct-treated mice to have lower tumor burdencompared to pure cytokine and predict that OMCP-R38 Å/F42K IL2 willdemonstrate the best efficacy for immunotherapy with the most favorableside effect profile.

If we do not see an effect of OMCP expressing IL2 constructs we willclosely evaluate our data for confounding factors such as excessive CTLdeath due to extreme stimulation as well as possible sequestration ofCTLs in systemic organs such as the liver and lungs. If our hypothesisis supported and NK cells are activated and tumor growth amelioratedafter OMCP-construct treatment we would repeat these experiments afterNK depletion (using anti-NK1.1 clone PK136, mouse anti-mouse depletingantibody) and CD8 depletion (clone YTS169, rat anti-mouse CD8⁺ Tcell-depleting antibody) (both from BioXcell, West Lebanon, N.H.). Basedon these results future work will focus on immunotherapy in primarycarcinogenesis models.

Example 16. The Effects of IL-2, R38 Å/F42K IL2 or OMCP Targeted IL2Constructs on Immunosuppression after Radiation Exposure

Sublethal radiation exposure is a constant risk to those involved incombat duty. In addition to the direct carcinogenic effects ofradiation-induced DNA damage, sublethal irradiation results inimmunologic damage due to selective death of lymphocyte subsets. CD8⁺ Tcells and CD44¹⁰ naïve T cells are specifically sensitive toradiation-induced death while NK cell function significantly declinesafter irradiation. CD4⁺25⁺ T cells as well as CD44 memory-like T cells,however, have a survival advantage after radiation. Both CD4⁺25⁺ T cellsand CD8⁺CD44^(hi) T cells can downregulate immune responses, explainingwhy even limited exposure to radiation can result in significantimmunosuppression. Pharmacologic interventions to restore the immunesystem can alleviate morbidity and mortality of radiation poisoning.Surprisingly the role of IL2 in alleviating radiation-induced changeshas never been studied. The low affinity IL2 receptor is expressed onbone marrow-resident hematopoietic stem cells and committed NKprogenitors. NK cells, in turn, can secrete granulocyte-macrophagecolony-stimulating factor (GM-CSF) upon stimulation, a cytokine that canassist with hematopoietic recovery. Based on these data in this aim weplan to test the hypothesis that IL2 or OMCP-IL2 constructs can assistwith hematopoietic recovery after sublethal and lethal irradiation.

Based on previously described models of radiation-induced hematopoieticdamage and recovery we will irradiate B6 mice with either sublethal 4.5or lethal 7.5Gy from a cesium source. Within one hour of exposure micein both radiation doses will be randomly divided into 13 groups asdescribed in Table 4 and treated for five days with low, intermediate orhigh dose IL2, R38 Å/F42K IL2 or OMCP expressing IL2 constructs (FIG.18). A portion of the mice will be injected with saline afterirradiation (group 13) (Table 4) and unirradiated untreated B6 mice willbe included as a control as well (group 14). On day 6 hematopoieticrecovery will be monitored by flow cytometric analysis of peripheralblood obtained by superficial mandibular vein sampling. The sample willbe analyzed for total number of NK cells, T cells, B cells,granulocytes, as well as monocytes and macrophages per ml of blood.Since 90% of untreated mice die 15-25 days after exposure to 7.5Gy, micewill be followed daily and survival curves in each treatment group willbe compared by Kaplan-Meier analysis. Moribund mice in the 7.5Gy groupwill be carefully analyzed for cause of death evaluating the bonemarrow, spleen and peripheral organs for both infection as well ashematopoietic failure by flow cytometry and tissue culture. Since micein the sublethal 4.5Gy group are expected to survive long term, theywill be sacrificed one month after exposure and peripheral lymphoidorgans as well as bone marrow evaluated for hematopoietic recovery byflow cytometric analysis.

Radiation related DNA damage results in malignant transformation.Hematopoietic malignancies are especially prominent after radiationexposure. In order to evaluate the ability of IL2 or OMCP linked IL2constructs to facilitate in clearing hematopoietic malignancies afterradiation exposure we will treat B6 mice with sublethal exposure to4.5Gy from a cesium source. Two days after irradiation the mice will beinjected with 103 RMA-S lymphoma cells i.p. and three days later treatedfor a five day course with low, intermediate or high dose IL2, R38Å/F42K IL2 or OMCP expressing IL2 constructs (Table 4, FIG. 19).Unirradiated B6 mice will be included as a control (group 14) as well.The mice will be followed for survival.

We anticipate that wild-type IL2 alone will have a negligible effect onimmunorestoration since it will most likely result in preferentialexpansion of CD4⁺Foxp3⁺ T_(regs), which are already preserved afterirradiation. We suspect, however, that R38 Å/F42K IL2 as well as OMCPexpressing IL2 constructs will expand the NK fraction in the peripheralblood and will contribute to broad hematopoietic recovery, albeitindirectly through secretion of homeostatic cytokines such as GM-CSF. Ifwe detect no differences in hematopoietic recovery between IL2 andsaline-treated groups, we will examine other confounding factors, suchas homeostatic proliferation induced alteration of the immune system andthe effect of IL2 or OMCP expressing IL2 constructs on suchproliferation. While 200,000 IU of IL2 administered daily to B6 mice isnot lethal, we realize that in the face of irradiation the mice might beweaker. It is thus possible that dosing might need to be adjusted. Forthe “functional” part of this experiment we plan to specifically utilizethe well-established model of RMA-S lymphoma challenge due to the roleof NK cells in controlling hematologic malignancies. This establishedassay will allow us to gain rapid experimental data to advance this aim.Based on this data we would extend this aim in the future utilizing aprimary carcinogenesis model as well.

Example 17. OMCP-Targeted Delivery of IL15 Enhances CD25 Upregulation

Interleukin 15 (IL15) is a cytokine with structural similarity to IL2.Like IL2, IL15 binds to and signals through a complex composed ofL2/IL15 receptor beta chain (CD122) and the common gamma chain (gamma-C,CD132). IL15 is secreted by mononuclear phagocytes (and some othercells) following infection by viruses. IL15 regulates T and naturalkiller (NK) cell activation and proliferation. Survival signals thatmaintain memory T cells in the absence of antigen are provided by IL15.This cytokine is also implicated in NK cell development. IL-15 belongsto the four α-helix bundle family of cytokine.

OMCP was linked to the cytokine IL15 and its ability to active NK cellscompared to IL15 alone was examined. NK cell activation was measured byCD25 upregulation. As demonstrated in FIG. 21, higher levels of CD25 areevident when IL15 is delivered by OMCP vs naked cytokine alone inequimolar doses.

Example 18. OMCP-Targeted Delivery of IL18 Enhances NK Cell Activation

OMCP was linked to WT human IL18, WT murine IL18 or mutant human IL18(which inhibits its interaction with IL18BP) and its ability to activeNK cells was examined (FIG. 32). Peripheral blood lymphocytes werecultured for 48 hours in 4.4 μM of either wild-type IL18 (blue),OMCP-IL18 (red) or saline (black). Activation of CD56+CD3− naturalkiller cells, as measured by surface CD69 expression, was superior byOMCP-IL18 compared to wild-type IL18 (FIG. 33). This data demonstratesthat linking OMCP to IL18 also enhances NK cell activation relative toIL18 without OMCP.

Example 19. The D132R Mutation in OMCP Significantly Decreases its NKG2DBinding

To further test the necessity of NKG2D binding in targeted delivery ofIL2, we tested NK expansion and activation in the presence of mutIL2,OMCP-mutIL2, and (D132R) OMCP-mutIL2. The D132R mutation ameliorated thesuperiority of natural killer cell activation over cytokine alone (FIG.22). Thus high affinity NKG2D binding is critical for targeted deliveryand lymphocyte activation by IL2.

Example 20. OMCP-IL2 Effectively Treats Infection Caused by West NileVirus (WNV)

The ability of various constructs of the invention to treat infectioncaused by West Nile Virus (WNV) was evaluated. Mice were given OMCP-IL2,the binding null mutant of OMCP, OMCP(D132R)-IL2, IL2 alone,IL2(38R/42A) alone and PBS. Upon treatment with OMCP(D132R)-IL2 and PBSall mice succumbed to infection by about day 11. Following treatmentwith IL2 alone, approximately 20% of mice survived until day 21.However, treatment with IL2(38R/42A) and OMCP-IL2 resulted in about 40%of mice surviving beyond 21 days (FIG. 30A). These results wereconsistently repeatable as demonstrated in FIG. 30B.

REFERENCES FOR EXAMPLE 11-20

-   1. Rosenberg, S. A. IL-2: the first effective immunotherapy for    human cancer. J Immunol 192, 5451-5458 (2014).-   2. Atkins, M. B., et al. High-dose recombinant interleukin 2 therapy    for patients with metastatic melanoma: analysis of 270 patients    treated between 1985 and 1993. J Clin Oncol 17, 2105-2116 (1999).-   3. Krieg, C., Letoumeau, S., Pantaleo, G. & Boyman, O. Improved IL-2    immunotherapy by selective stimulation of IL-2 receptors on    lymphocytes and endothelial cells. Proc Natl Acad Sci USA 107,    11906-11911 (2010).-   4. Ghiringhelli, F., Menard, C., Martin, F. & Zitvogel, L. The role    of regulatory T cells in the control of natural killer cells:    relevance during tumor progression. Immunol Rev 214, 229-238 (2006).-   5. French, A. R., et al. DAP12 signaling directly augments    proproliferative cytokine stimulation of NK cells during viral    infections. J Immunol 177, 4981-4990 (2006).-   6. Heaton, K. M., Ju, G. & Grimm, E. A. Human interleukin 2    analogues that preferentially bind the intermediate-affinity    interleukin 2 receptor lead to reduced secondary cytokine secretion:    implications for the use of these interleukin 2 analogues in cancer    immunotherapy. Cancer Res 53, 2597-2602 (1993).-   7. Heaton, K. M., et al. Characterization of lymphokine-activated    killing by human peripheral blood mononuclear cells stimulated with    interleukin 2 (IL-2) analogs specific for the intermediate affinity    IL-2 receptor. Cellular immunology 147, 167-179 (1993).-   8. Levin, A. M., et al. Exploiting a natural conformational switch    to engineer an interleukin-2 ‘superkine’. Nature 484, 529-533    (2012).-   9. Campbell, J. A., Trossman, D. S., Yokoyama, W. M. &    Carayannopoulos, L. N. Zoonotic orthopoxviruses encode a    high-affinity antagonist of NKG2D. J Exp Med 204, 1311-1317 (2007).-   10. Rosenberg, S. A., et al. Experience with the use of high-dose    interleukin-2 in the treatment of 652 cancer patients. Annals of    surgery 210, 474-484; discussion 484-475 (1989).-   11. Gately, M. K., Anderson, T. D. & Hayes, T. J. Role of    asialo-GM1-positive lymphoid cells in mediating the toxic effects of    recombinant IL-2 in mice. J Immunol 141, 189-200 (1988).-   12. Sim, G. C., et al. IL-2 therapy promotes suppressive ICOS+ Treg    expansion in melanoma patients. J Clin Invest 124, 99-110 (2014).-   13. Raulet, D. H. Roles of the NKG2D immunoreceptor and its ligands.    Nat Rev Immunol 3, 781-790 (2003).-   14. Ullrich, E., Koch, J., Cerwenka, A. & Steinle, A. New prospects    on the NKG2D/NKG2DL system for oncology. Oncoimmunology 2, e26097    (2013).-   15. Lazear, E., Peterson, L. W., Nelson, C. A. & Fremont, D. H.    Crystal structure of the cowpox virus-encoded NKG2D ligand OMCP. J    Virol 87, 840-850 (2013).-   16. Bui, J. D., Uppaluri, R., Hsieh, C. S. & Schreiber, R. D.    Comparative analysis of regulatory and effector T cells in    progressively growing versus rejecting tumors of similar origins.    Cancer Res 66, 7301-7309 (2006).-   17. Wang, Y., Sparwasser, T., Figlin, R. & Kim, H. L. Foxp3+ T cells    inhibit antitumor immune memory modulated by mTOR inhibition. Cancer    Res 74, 2217-2228 (2014).-   18. Poschke, I., et al. A phase I clinical trial combining dendritic    cell vaccination with adoptive T cell transfer in patients with    stage IV melanoma. Cancer immunology, immunotherapy: CII63,    1061-1071 (2014).-   19. Kreisel, D., et al. Strain-specific variation in murine natural    killer gene complex contributes to differences in immunosurveillance    for urethane-induced lung cancer. Cancer Res 72, 4311-4317 (2012).-   20. Frese-Schaper, M., et al. Influence of natural killer cells and    perforinmediated cytolysis on the development of chemically induced    lung cancer in A/J mice. Cancer immunology, immunotherapy: CII63,    571-580 (2014).-   21. Ryu, M. S., et al. Accumulation of cytolytic CD8(+) T cells in    B16-melanoma and proliferation of mature T cells in TIS21-knockout    mice after T cell receptor stimulation. Experimental cell research    327, 209221 (2014).-   22. Anichini, A., et al. Tumor-reactive CD8+ early effector T cells    identified at tumor site in primary and metastatic melanoma. Cancer    Res 70, 8378-8387 (2010).-   23. Glasner, A., et al. Recognition and prevention of tumor    metastasis by the NK receptor NKp46/NCR1. J Immunol 188, 2509-2515    (2012).-   24. Hersey, P., Edwards, A., Honeyman, M. & McCarthy, W. H. Low    natural-killer-cell activity in familial melanoma patients and their    relatives. Br J Cancer 40, 113-122 (1979).-   25. Ji, Q., Gondek, D. & Hurwitz, A. A. Provision of    granulocyte-macrophage colony-stimulating factor converts an    autoimmune response to a self-antigen into an antitumor response. J    Immunol 175, 14561463 (2005).-   26. Zhu, Z., et al. High-avidity T cells are preferentially    tolerized in the tumor microenvironment. Cancer Res 73, 595-604    (2013).-   27. Klein, O., et al. Melan-A-specific cytotoxic T cells are    associated with tumor regression and autoimmunity following    treatment with anti-CTLA-4. Clinical cancer research: an official    journal of the American Association for Cancer Research 15,    2507-2513 (2009).-   28. Meiraz, A., Garber, O. G., Harari, S., Hassin, D. & Berke, G.    Switch from perforin-expressing to perforin-deficient CD8(+) T cells    accounts for two distinct types of effector cytotoxic T lymphocytes    in vivo. Immunology 128, 69-82 (2009).-   29. Stemberger, C., et al. A single naive CD8+ T cell precursor can    develop into diverse effector and memory subsets. Immunity 27,    985-997 (2007).-   30. Sallusto, F., Lenig, D., Forster, R., Lipp, M. &    Lanzavecchia, A. Two subsets of memory T lymphocytes with distinct    homing potentials and effector functions. Nature 401, 708-712    (1999).-   31. Araki, K., et al. mTOR regulates memory CD8 T-cell    differentiation. Nature 460, 108-112 (2009).-   32. Kim, H. L. Antibody-based depletion of Foxp3+ T cells    potentiates antitumor immune memory stimulated by mTOR inhibition.    Oncoimmunology 3, e29081 (2014).-   33. Graham, J. B., Da Costa, A. & Lund, J. M. Regulatory T cells    shape the resident memory T cell response to virus infection in the    tissues. J Immunol 192, 683-690 (2014).-   34. de Goer de Herve, M. G., Jaafoura, S., Vallee, M. & Taoufik, Y.    FoxP3(+) regulatory CD4 T cells control the generation of functional    CD8 memory. Nature communications 3, 986 (2012).-   35. Gilfillan, S., Ho, E. L., Cella, M., Yokoyama, W. M. &    Colonna, M. NKG2D recruits two distinct adapters to trigger NK cell    activation and costimulation. Nature immunology 3, 1150-1155 (2002).-   36. Shane, H. L. & Klonowski, K. D. Every breath you take: the    impact of environment on resident memory CD8 T cells in the lung.    Frontiers in immunology 5, 320 (2014).-   37. Marcus, A. & Raulet, D. H. Evidence for natural killer cell    memory. Current biology: CB 23, R817-820 (2013).-   38. Tam, S. H., Sassoli, P. M., Jordan, R. E. & Nakada, M. T.    Abciximab (ReoPro, chimeric 7E3 Fab) demonstrates equivalent    affinity and functional blockade of glycoprotein IIb/IIIa and    alpha(v)beta3 integrins. Circulation 98, 1085-1091 (1998).-   39. Trikha, M., et al. CNTO 95, a fully human monoclonal antibody    that inhibits alphav integrins, has antitumor and antiangiogenic    activity in vivo. Internationaljournal of cancer. Journal    intemational du cancer 110, 326-335 (2004).-   40. Rathanaswami, P., Babcook, J. & Gallo, M. High-affinity binding    measurements of antibodies to cell-surface-expressed antigens. Anal    Biochem 373, 52-60 (2008).-   41. Drake, A. W., Myszka, D. G. & Klakamp, S. L. Characterizing    high-affinity antigen/antibody complexes by kinetic- and    equilibrium-based methods. Anal Biochem 328, 35-43 (2004).-   42. Siiman, O. & Burshteyn, A. Cell surface receptor-antibody    association constants and enumeration of receptor sites for    monoclonal antibodies. Cytometry 40, 316-326 (2000).-   43. Debbia, M. & Lambin, P. Measurement of anti-D intrinsic affinity    with unlabeled antibodies. Transfusion 44, 399-406 (2004).-   44. Tsao, P. I. & von Zastrow, M. Type-specific sorting of G    protein-coupled receptors after endocytosis. The Journal of    biological chemistry 275, 11130-11140 (2000).-   45. Lazear, E., et al. Cowpox virus OMCP antagonizes NKG2D via an    unexpected binding orientation. PLos Pathogen Under review(2014).-   46. Liu, K. D., Gaffen, S. L., Goldsmith, M. A. & Greene, W. C.    Janus kinases in interleukin-2-mediated signaling: JAK1 and JAK3 are    differentially regulated by tyrosine phosphorylation. Current    biology: CB 7, 817-826 (1997).-   47. Zhou, Y. J., et al. Distinct tyrosine phosphorylation sites in    JAK3 kinase domain positively and negatively regulate its enzymatic    activity. Proc Natl Acad Sci USA 94, 13850-13855 (1997).-   48. Homg, T., Bezbradica, J. S. & Medzhitov, R. NKG2D signaling is    coupled to the interleukin 15 receptor signaling pathway. Nature    immunology 8, 1345-1352 (2007).-   49. Zou, W., Reeve, J. L., Liu, Y., Teitelbaum, S. L. & Ross, F. P.    DAP12 couples c-Fms activation to the osteoclast cytoskeleton by    recruitment of Syk. Molecular cell 31, 422-431 (2008).-   50. Graham, D. B., et al. Vav1 controls DAP10-mediated natural    cytotoxicity by regulating actin and microtubule dynamics. J Immunol    177, 2349-2355 (2006).-   51. Yamane, B. H., Hank, J. A., Albertini, M. R. & Sondel, P. M. The    development of antibody-IL-2 based immunotherapy with hul4.18-IL2    (EMD-273063) in melanoma and neuroblastoma. Expert opinion on    investigational drugs 18, 991-1000 (2009).-   52. Becker, J. C., Pancook, J. D., Gillies, S. D., Furukawa, K. &    Reisfeld, R. A. T cell-mediated eradication of murine metastatic    melanoma induced by targeted interleukin 2 therapy. J Exp Med 183,    2361-2366 (1996).-   53. Lundholm et al., Prostate tumor-derived exosomes down-regulate    NKG2D expression on natural killer cells and CD8+ T cells: mechanism    of immune evasion. PLoS One 2014; 9(9):e108925.

Lengthy table referenced here US20210032306A1-20210204-T00001 Pleaserefer to the end of the specification for access instructions.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210032306A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A chimeric peptide comprising a first peptidelinked to a second peptide, wherein the first peptide specifically bindsto NKG2D receptor and the second peptide comprises an amino acidsequence of at least 80% homology to SEQ ID NO: 5, wherein the secondpeptide has a binding affinity to the IL2βγ receptor subunit that issimilar to or greater than a peptide comprising an amino acid sequenceof 100% homology to SEQ ID NO:
 5. 2. The chimeric peptide of claim 1,wherein the first peptide comprises an amino acid sequence of at least50% homology to SEQ ID NO: 7 and has a binding affinity to the NKG2Dreceptor of from about 0.1 nM to about 100 nM.